Antibodies that bind to the C35 polypeptide

ABSTRACT

The present invention relates to a novel human gene that is differentially expressed in human carcinoma. More specifically, the present invention relates to a polynucleotide encoding a novel human polypeptide named C35 that is overexpressed in human breast and bladder carcinoma. This invention also relates to C35 polypeptide, in particular C35 peptide epitopes and C35 peptide epitope analogs, as well as vectors, host cells, antibodies directed to C35 polypeptides, and the recombinant methods for producing the same. The present invention further relates to diagnostic methods for detecting carcinomas, including human breast carcinomas. The present invention further relates to the formulation and use of the C35 gene and polypeptides, in particular C35 peptide epitopes and C35 peptide epitope analogs, in immunogenic compositions or vaccines, to induce antibody or cell-mediated immunity against target cells, such as tumor cells, that express the C35 gene. The invention further relates to screening methods for identifying agonists and antagonists of C35 activity.

This application is a divisional of U.S. patent application Ser. No.10/457,829, now U.S. Pat. No. 7,563,882, filed Jun. 10, 2003, whichclaims the benefit of U.S. Provisional Application No. 60/386,738, filedJun. 10, 2002, and U.S. Provisional Application No. 60/432,241, filedDec. 11, 2002, and U.S. Provisional Application No. 60/464,650, filedApr. 23, 2003, the entire contents of which are hereby incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Part of the work performed during development of this invention utilizedU.S. Government funds. The U.S. Government has certain rights in thisinvention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

This application includes a “Sequence Listing,”“substitute_sequence_listing_ascii.txt”, 1,105,341 bytes, created onNov. 1, 2010 and submitted electronically via EFS-Web which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a novel human gene that isdifferentially expressed in human breast and bladder carcinoma. Morespecifically, the present invention relates to a polynucleotide encodinga novel human polypeptide named C35. This invention also relates to C35polypeptides, as well as vectors, host cells, antibodies directed to C35polypeptides, and the recombinant methods for producing the same. Thepresent invention further relates to diagnostic methods for detectingcarcinomas, including human breast and bladder carcinomas. The presentinvention further relates to the formulation and use of the C35 gene andpolypeptides in immunogenic compositions or vaccines, to induce antibodyand cell-mediated immunity against target cells, such as tumor cells,that express the C35 gene. The invention further relates to screeningmethods for identifying agonists and antagonists of C35 activity.

BACKGROUND ART

Cancer afflicts approximately 1.2 million people in the United Stateseach year. About 50% of these cancers are curable with surgery,radiation therapy, and chemotherapy. Despite significant technicaladvances in these three types of treatments, each year more than 500,000people will die of cancer in the United States alone. (Jaffee, E. M.,Ann. N.Y. Acad. Sci. 886:67-72 (1999)). Because most recurrences are atdistant sites such as the liver, brain, bone, and lung, there is anurgent need for improved systemic therapies.

The goal of cancer treatment is to develop modalities that specificallytarget tumor cells, thereby avoiding unnecessary side effects to normaltissue. Immunotherapy has the potential to provide an alternativesystemic treatment for most types of cancer. The advantage ofimmunotherapy over radiation and chemotherapy is that it can actspecifically against the tumor without causing normal tissue damage. Oneform of immunotherapy, vaccines, is particularly attractive because theycan also provide for active immunization, which allows for amplificationof the immune response. In addition, vaccines can generate a memoryimmune response.

The possibility that altered features of a tumor cell are recognized bythe immune system as non-self and may induce protective immunity is thebasis for attempts to develop cancer vaccines. Whether or not this is aviable strategy depends on how the features of a transformed cell arealtered. Appreciation of the central role of mutation in tumortransformation gave rise to the hypothesis that tumor antigens arise asa result of random mutation in genetically unstable cells. Althoughrandom mutations might prove immunogenic, it would be predicted thatthese would induce specific immunity unique for each tumor. This wouldbe unfavorable for development of broadly effective tumor vaccines. Analternate hypothesis, however, is that a tumor antigen may arise as aresult of systematic and reproducible tissue specific gene deregulationthat is associated with the transformation process. This could give riseto qualitatively or quantitatively different expression of sharedantigens in certain types of tumors that might be suitable targets forimmunotherapy. Early results, demonstrating that the immunogenicity ofsome experimental tumors could be traced to random mutations (De Plaen,et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988); Srivastava, &Old, Immunol. Today 9:78 (1989)), clearly supported the firsthypothesis. There is, however, no a priori reason why random mutationand systematic gene deregulation could not both give rise to newimmunogenic expression in tumors. Indeed, more recent studies in bothexperimental tumors (Sahasrabudhe et al., J. Immunol. 151:6202-6310(1993); Torigoe et al., J. Immunol. 147:3251 (1991)) and human melanoma(van Der Bruggen et al., Science 254:1643-1647 (1991); Brichard et al.,J. Exp. Med. 178:489-495 (1993); Kawakami et al., Proc. Natl. Acad. Sci.USA 91:3515-3519 (1994); Boel et al., Immunity 2:167-175 (1995); Van denEynde et al., J. Exp. Med. 182: 689-698 (1995)) have clearlydemonstrated expression of shared tumor antigens encoded by deregulatednormal genes. The identification of MAGE-1 and other antigens common todifferent human melanoma holds great promise for the future developmentof multiple tumor vaccines.

In spite of the progress in melanoma, very few shared antigensrecognized by cytotoxic T cells have not been described for other humantumors. The major challenge is technological. The most widespread and todate most successful approach to identify immunogenic molecules uniquelyexpressed in tumor cells is to screen a cDNA library with tumor-specificCTLs (cytotoxic T lymphocytes). Application of this strategy has led toidentification of several gene families expressed predominantly in humanmelanoma. Two major limitations of this approach, however, are that (1)screening requires labor intensive transfection of numerous small poolsof recombinant DNA into separate target populations, which themselvesoften need to be modified to express one or more MHC molecules requiredfor antigen presentation, in order to assay T cell stimulation by aminor component of some pool; and (2) with the possible exception ofrenal cell carcinoma, tumor-specific CTLs have been very difficult toisolate from either tumor infiltrating lymphocytes (TIL) or PBL ofpatients with other types of tumors, especially the epithelial cellcarcinomas that comprise greater than 80% of human tumors. It appearsthat there may be tissue specific properties that result intumor-specific CTLs being sequestered in melanoma.

Direct immunization with tumor-specific gene products may be essentialto elicit an immune response against some shared tumor antigens. It hasbeen argued that, if a tumor expressed strong antigens, it should havebeen eradicated prior to clinical manifestation. Perhaps then, tumorsexpress only weak antigens. Immunologists have long been interested inthe issue of what makes an antigen weak or strong. There have been twomajor hypotheses. Weak antigens may be poorly processed and fail to bepresented effectively to T cells. Alternatively, the number of T cellsin the organism with appropriate specificity might be inadequate for avigorous response (a so-called “hole in the repertoire”). Elucidation ofthe complex cellular process whereby antigenic peptides associate withMHC molecules for transport to the cell surface and presentation to Tcells has been one of the triumphs of modern immunology. Theseexperiments have clearly established that failure of presentation due toprocessing defects or competition from other peptides could render aparticular peptide less immunogenic. In contrast, it has, for technicalreasons, been more difficult to establish that the frequency of clonalrepresentation in the T cell repertoire is an important mechanism of lowresponsiveness. Recent studies demonstrating that the relationshipbetween immunodominant and cryptic peptides of a protein antigen changein T cell receptor transgenic mice suggest, however, that the relativefrequency of peptide-specific T cells can, indeed, be a determiningfactor in whether a particular peptide is cryptic or dominant in a Tcell response. This has encouraging implications for development ofvaccines. With present day methods, it would be a complex and difficultundertaking to modify the way in which antigenic peptides of a tumor areprocessed and presented to T cells. The relative frequency of a specificT cell population can, however, be directly and effectively increased byprior vaccination. This could, therefore, be the key manipulationrequired to render an otherwise cryptic response immunoprotective. Theseconsiderations of cryptic or sub-dominant antigens have specialrelevance in relation to possible immune evasion by tumors throughtolerance induction. Evidence has been presented to suggest thattumor-specific T cells in the tumor-bearing host are anergic, possiblyas a result of antigen presentation on non-professional APC (Morgan, D.J. et al., J. Immunol. 163:723-27 (1999); Sotomayor, E. M. et al., Proc.Natl. Acad. Sci. U.S.A. 96:11476-81 (1999); Lee, P. P. et al., NatureMedicine 5:677-85 (1999)). Prior tolerization of T cells specific forimmunodominant antigens of a tumor may, therefore, account for thedifficulty in developing successful strategies for immunotherapy ofcancer. These observations suggest that T cells specific forimmunodominant tumor antigens are less likely to be effective forimmunotherapy of established tumors because they are most likely to havebeen tolerized. It may, therefore, be that T cells specific forsub-dominant antigens or T cells that are initially present at a lowerfrequency would prove more effective because they have escaped thetolerizing influence of a growing tumor.

Another major concern for the development of broadly effective humanvaccines is the extreme polymorphism of HLA class I molecules. Class IMHC:cellular peptide complexes are the target antigens for specific CD8+CTLs. The cellular peptides, derived by degradation of endogenouslysynthesized proteins, are translocated into a pre-Golgi compartmentwhere they bind to class I MHC molecules for transport to the cellsurface. The CD8 molecule contributes to the avidity of the interactionbetween T cell and target by binding to the α3 domain of the class Iheavy chain. Since all endogenous proteins turn over, peptides derivedfrom any cytoplasmic or nuclear protein may bind to an MHC molecule andbe transported for presentation at the cell surface. This allows T cellsto survey a much larger representation of cellular proteins thanantibodies which are restricted to recognize conformational determinantsof only those proteins that are either secreted or integrated at thecell membrane.

The T cell receptor antigen binding site interacts with determinants ofboth the peptide and the surrounding MHC. T cell specificity must,therefore, be defined in terms of an MHC:peptide complex. Thespecificity of peptide binding to MHC molecules is very broad and ofrelatively low affinity in comparison to the antigen binding sites ofspecific antibodies. Class I-bound peptides are generally 8-10 residuesin length and accommodate amino acid side chains of restricted diversityat certain key positions that match pockets in the MHC peptide bindingsite. These key features of peptides that bind to a particular MHCmolecule constitute a peptide binding motif.

Hence, there exists a need for methods to facilitate the induction andisolation of T cells specific for human tumors, cancers and infectedcells and for methods to efficiently select the genes that encode themajor target antigens recognized by these T cells in the properMHC-context.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a novel polynucleotide, C35, that isdifferentially expressed in human breast and bladder carcinoma, and tothe encoded polypeptide of C35. Moreover, the present invention relatesto vectors, host cells, antibodies, and recombinant methods forproducing C35 polypeptides and polynucleotides. The present inventionfurther relates to the formulation and use of C35 polypeptides, inparticular C35 peptide epitopes and C35 peptide epitope analogs, andpolynucleotides in immunogenic compositions to induce antibodies andcell-mediated immunity against target cells, such as tumor cells, thatexpress the C35 gene products. Also provided are diagnostic methods fordetecting disorders relating to the C35 genes and polypeptides,including use as a prognostic marker for carcinomas, such as humanbreast carcinoma, and therapeutic methods for treating such disorders.The invention further relates to screening methods for identifyingbinding partners of C35.

Thus, in one embodiment, the invention relates to an isolatedpolypeptide comprising a peptide comprising two or more C35 peptideepitopes, wherein said peptide is selected from the group consisting of:amino acids T101 to V113 of SEQ ID NO:2, E100 to V113 of SEQ ID NO:2,G99 to V113 of SEQ ID NO:2, I93 to V113 of SEQ ID NO:2, D88 to V113 ofSEQ ID NO:2, P84 to V113 of SEQ ID NO:2, K77 to V113 of SEQ ID NO:2, Q72to V113 of SEQ ID NO:2, F65 to V113 of SEQ ID NO:2, and L59 to V113 ofSEQ ID NO:2, and wherein said isolated polypeptide is not SEQ ID NO: 2,SEQ ID NO: 153, SEQ ID NO: 155, or amino acids E100 to R109 of SEQ IDNO:2.

In another embodiment, the invention relates to an isolated polypeptidecomprising at least one C35 peptide epitope analog, wherein said C35peptide epitope analog is selected from the group consisting of: for thepeptide epitope G22 to C30 of SEQ ID NO:2 and FIG. 1B, the analogs witheither alanine or glycine substituted for cysteine at the ninth aminoacid residue; for the peptide epitope I25 to C33 of SEQ ID NO:2 and FIG.1B, the analogs with either alanine or glycine substituted for thecysteine at the sixth amino acid residue and/or the ninth amino acidresidue; for the peptide epitope K77 to Y85 of SEQ ID NO: 2 and FIG. 1B,the analog with valine substituted for tyrosine at the ninth amino acidresidue; for the peptide epitope K104 to C112 of SEQ ID NO:2 and FIG.1B, the analogs with alanine, glycine or leucine substituted forcysteine at the ninth amino acid residue; for the peptide epitope K104to V113 of SEQ ID NO:2 and FIG. 1B, the analogs with alanine, serine,glycine or leucine substituted for cysteine at the ninth amino acidresidue; for the peptide epitope I105 to V113 of SEQ ID NO:2 and FIG.1B, the analogs with either leucine or methionine substituted forthreonine at the second amino acid residue and/or alanine, serine orglycine substituted for cysteine at the eighth amino acid residue; andfor the peptide epitope N107 to L115 of SEQ ID NO:2 and FIG. 1B, theanalog with either alanine or glycine substituted for cysteine at thesixth amino acid residue.

Preferably the isolated polypeptide of the invention is not more than100 amino acids in length, alternatively not more that 95 amino acids inlength, alternatively not more than 90 amino acids in length,alternatively not more than 85 amino acids in length, alternatively notmore than 80 amino acids in length, alternatively not more than 75 aminoacids in length, alternatively not more than 70 amino acids in length,alternatively not more than 65 amino acids in length, alternatively notmore than 60 amino acids in length, alternatively not more than 55 aminoacids in length, alternatively not more than 50 amino acids in length,alternatively not more than 45 amino acids in length, alternatively notmore than 40 amino acids in length, or alternatively not more than 35amino acids in length.

In another embodiment, the invention relates to a fusion proteincomprising an isolated peptide comprising two or more C35 peptideepitopes, wherein said isolated peptide is selected from the groupconsisting of: amino acids T101 to V113 of SEQ ID NO:2, E100 to V113 ofSEQ ID NO:2, G99 to V113 of SEQ ID NO:2, I93 to V113 of SEQ ID NO:2, D88to V113 of SEQ ID NO:2, P84 to V113 of SEQ ID NO:2, K77 to V113 of SEQID NO:2, Q72 to V113 of SEQ ID NO:2, F65 to V113 of SEQ ID NO:2, and L59to V113 of SEQ ID NO:2. In a preferred embodiment, the fusion protein isa homopolymer of said isolated peptide. In another preferred embodiment,the fusion protein is a heteropolymer of said isolated polypeptides. Inyet another embodiment, the fusion protein is fused to a T helperpeptide. In still another embodiment, the fusion protein is fused to acarrier. In another embodiment, the fusion protein is linked to a lipid.

In another embodiment, the invention relates to an isolated polypeptideconsisting of two or more C35 peptide epitopes, wherein said isolatedpolypeptide is selected from the group consisting of: amino acids T101to V113 of SEQ ID NO:2, E100 to V113 of SEQ ID NO:2, G99 to V113 of SEQID NO:2, I93 to V113 of SEQ ID NO:2, D88 to V113 of SEQ ID NO:2, P84 toV113 of SEQ ID NO:2, K77 to V113 of SEQ ID NO:2, Q72 to V113 of SEQ IDNO:2, F65 to V113 of SEQ ID NO:2, and L59 to V113 of SEQ ID NO:2, andwherein said isolated polypeptide is not SEQ ID NO: 2, SEQ ID NO: 153,SEQ ID NO: 155, or amino acids E100 to R109 of SEQ ID NO:2.

In another embodiment, the invention relates to an isolated polypeptidecomprising a peptide comprising at least one C35 peptide epitope analog,wherein said peptide is selected from the group consisting of the analogof peptide T101 to V113 of SEQ ID NO:2 having either alanine or glycinesubstituted for the cysteine at the twelfth residue, the analog ofpeptide E100 to V113 of SEQ ID NO:2 having either alanine or glycinesubstituted for the cysteine at the thirteenth residue, the analog ofpeptide G99 to V113 of SEQ ID NO:2 having either alanine or glycinesubstituted for cysteine at the fourteenth residue, the analog ofpeptide I93 to V113 of SEQ ID NO:2 having either alanine or glycinesubstituted for the cysteine at the twentieth residue, the analog ofpeptide D88 to V113 of SEQ ID NO:2 having either alanine or glycinesubstituted for the cysteine at the twenty-fifth residue, the analog ofpeptide P84 to V113 of SEQ ID NO:2 having either alanine or glycinesubstituted for the cysteine at the twenty-ninth residue, the analog ofpeptide K77 to V113 of SEQ ID NO:2 having either alanine or glycinesubstituted for the cysteine at the thirty-sixth residue, the analog ofpeptide Q72 to V113 of SEQ ID NO:2 having either alanine or glycinesubstituted for the cysteine at the forty-first residue, the analog ofpeptide F65 to V113 of SEQ ID NO:2 having either alanine or glycinesubstituted for the cysteine at the forty-eighth residue, and the analogof peptide L59 to V113 of SEQ ID NO:2 having either alanine or glycinesubstituted for the cysteine at the fifty-fourth residue.

In another embodiment, the invention relates to a fusion proteincomprising a peptide comprising at least one C35 peptide epitope analog,wherein said peptide is selected from the group consisting of: for thepeptide epitope G22 to C30 of SEQ ID NO:2 and FIG. 1B, the analogs witheither alanine or glycine substituted for cysteine at the ninth aminoacid residue; for the peptide epitope I25 to C33 of SEQ ID NO:2 and FIG.1B, the analogs with either alanine or glycine substituted for thecysteine at the sixth amino acid residue and/or the ninth amino acidresidue; for the peptide epitope K77 to Y85 of SEQ ID NO: 2 and FIG. 1B,the analog with valine substituted for tyrosine at the ninth amino acidresidue; for the peptide epitope K104 to C112 of SEQ ID NO:2 and FIG.1B, the analogs with alanine, glycine or leucine substituted forcysteine at the ninth amino acid residue; for the peptide epitope K104to V113 of SEQ ID NO:2 and FIG. 1B, the analogs with alanine, glycine,serine or leucine substituted for cysteine at the ninth amino acidresidue; for the peptide epitope I105 to V113 of SEQ ID NO:2 and FIG.1B, the analogs with either leucine, serine or methionine substitutedfor threonine at the second amino acid residue and/or alanine or glycinesubstituted for cysteine at the eighth amino acid residue; and for thepeptide epitope N107 to V113 of SEQ ID NO:2 and FIG. 1B, the analog witheither alanine or glycine substituted for cysteine at the sixth aminoacid residue, the analog of peptide T101 to V113 of SEQ ID NO:2 havingeither alanine or glycine substituted for the cysteine at the twelfthresidue, the analog of peptide E100 to V113 of SEQ ID NO:2 having eitheralanine or glycine substituted for cysteine at the thirteenth residue,the analog of peptide G99 to V113 of SEQ ID NO:2 having either alanineor glycine substituted for cysteine at the fourteenth residue, theanalog of peptide I93 to V113 of SEQ ID NO:2 having either alanine orglycine substituted for the cysteine at the twentieth residue, theanalog of peptide D88 to V113 of SEQ ID NO:2 having either alanine orglycine substituted for the cysteine at the twenty-fifth residue, theanalog of peptide P84 to V113 of SEQ ID NO:2 having either alanine orglycine substituted for the cysteine at the twenty-ninth residue, theanalog of peptide K77 to V113 of SEQ ID NO:2 having either alanine orglycine substituted for the cysteine at the thirty-sixth residue, theanalog of peptide Q72 to V113 of SEQ ID NO:2 having either alanine orglycine substituted for the cysteine at the forty-first residue, theanalog of peptide F65 to V113 of SEQ ID NO:2 having either alanine orglycine substituted for the cysteine at the forty-eighth residue, andthe analog of peptide L59 to V113 of SEQ ID NO:2 having either alanineor glycine substituted for the cysteine at the fifty-fourth residue. Ina preferred embodiment, the fusion protein comprises a homopolymer ofsaid peptide comprising at least one C35 peptide epitope analog. Inanother preferred embodiment, the fusion protein comprises aheteropolymer of said peptide comprising at least one C35 peptideepitope analog.

In another embodiment, the invention relates to a composition comprisingan isolated polypeptide or fusion protein of the invention and apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B. FIG. 1A shows the DNA coding sequence (SEQ ID NO: 1) ofC35. The sequence immediately upstream of the predicted ATG start codonis shown in lower case and conforms to the expected features describedby Kozak, M., J. Biol. Chem. 266(30):19867-19870 (1991). FIG. 1B showsthe deduced amino acid sequence (SEQ ID NO: 2) of C35. The asterisk(“*”) represents a termination codon and signifies the end of theprotein sequence.

FIGS. 2A-2C. FIG. 2A: C35 is overexpressed in Breast tumor cell lines.Upper Panel: 300 ng of poly-A RNA from 3 week old human thymus, normalbreast epithelial cell line H16N2 from patient 21, and 4 breast tumorcell lines derived one year apart from primary or metastatic nodules ofthe same patient 21; 21NT, 21PT 21MT1, and 21MT2, was resolved on a 1%agarose/formaldehyde gel and transferred to a GENESCREEN™ membrane. Thisblot was hybridized with a ³²P labeled C35 probe. Hybridization wasdetected by exposing the blot to film for 15 hours. Lower Panel: Toquantitate RNA loading, the same blot was stripped and re-hybridizedwith a ³²P labeled probe for Glyceraldehyde-3 Phosphate Dehydrogenase(GAPDH). For each sample the C35 signal was normalized to the GAPDHsignal. The numbers represent the fold expression of C35 in each samplerelative to H16N2. FIG. 2B: C35 is expressed at low levels in normaltissues. A Blot containing 1 microgram of poly-A RNA from each of theindicated adult normal tissues (Clontech) was hybridized with a ³²Plabeled C35 probe. Hybridization was detected by exposing the blot tofilm for 15 hours (upper panel), or 96 hours (lower panel). FIG. 2C. C35is overexpressed in primary Breast tumors. A blot containing 2micrograms of poly-A RNA from 3 primary infiltrating ductal mammarycarcinoma, T1, T2, T3 and 1 normal breast epithelium, N (Invitrogen) washybridized with a ³²P labeled C35 probe. To normalize loading a ³²Plabeled beta-Actin probe was included in the hybridization mix.Hybridization was detected by exposing the blot to film for 6 hours. Thenumbers represent the fold expression of C35 in each sample relative tonormal breast epithelium.

FIG. 3. Expression of C35 in Breast Tumor Cell Lines. C35 isoverexpressed in different breast tumor cell lines. Upper Panel: 300 ngof poly-A RNA from BT474 (ATCC HYB-20, mammary ductal carcinoma), SKBR3(ATCC HTB-30, mammary adenocarcinoma), T47D (ATCC HTB-133, mammaryductal carcinoma), normal breast epithelial cell line H16N2 from patient21, and 21-NT breast tumor cell line derived from primary tumor noduleof the same patient 21 was resolved on a 1% agarose/formaldehyde gel andtransferred to a GENESCREEN™ membrane. This blot was hybridized with a³²P labeled C35 probe. Hybridization was detected by exposing the blotto film for 15 hours. Lower Panel: To quantitate RNA loading, the sameblot was stripped and re-hybridized with a ³²P labeled probe forbeta-actin. For each sample the C35 signal was normalized to the actinsignal. The numbers represent the fold expression of C35 in each samplerelative to H16N2.

FIGS. 4A-4C. Surface Expression of C35 Protein Detected by FlowCytometry. 1×10⁵ breast tumor cells were stained with 3.5 microliters ofantiserum raised in BALB/c mice against Line 1 mouse tumor cellstransduced with retrovirus encoding human C35 or control, pre-bleedBALB/c serum. After a 30 minute incubation, cells were washed twice withstaining buffer (PAB) and incubated with FITC-goat anti-mouse IgG (1ug/sample) for 30 minutes. Samples were washed and analyzed on an EPICSElite flow cytometer. FIG. 4A: 21NT. FIG. 4B: SKBR3. FIG. 4C:MDA-MB-231. These three breast tumor lines were selected to representtumor cells that express high, intermediate and low levels of C35 RNA onNorthern blots (see FIG. 3). Abbreviations: nms, ns; normal mouse serum;C35; C35 immune serum.

FIGS. 5A and 5B. CML Selected Recombinant Vaccinia cDNA Clones StimulateTumor Specific CTL. FIG. 5A: CML Selected vaccinia clones were assayedfor the ability, following infection of B/C.N, to stimulate tumorspecific CTL to secrete interferon gamma. The amount of cytokine wasmeasured by ELISA, and is represented as OD490 (14). An OD490 of 1.4 isapproximately equal to 4 ng/ml of IFNg, and an OD490 of 0.65 isapproximately equal to 1 ng/ml of IFNg. FIG. 5B: CML selected clonessensitize host cells to lysis by tumor specific CTL. Monolayers of B/C.Nin wells of a 6 well plate were infected with moi=1 of the indicatedvaccinia virus clones. After 14 hours of infection the infected cellswere harvested and along with the indicated control targets labeled with⁵¹Cr. Target cells were incubated with the indicated ratios of tumorspecific Cytotoxic T Lymphocytes for 4 hours at 37° C. and percentagespecific lysis was determined (15). This experiment was repeated atleast three times with similar results.

FIGS. 6A and 6B. The Tumor Antigen Is Encoded by a Ribosomal Protein L3Gene. Sequence of H2.16 and rpL3 from amino acid position 45 to 56. FIG.6A: The amino acid (in single letter code) (SEQ ID NO: 2143) andnucleotide sequence of cDNA clone rpL3 (GenBank Accession no. Y00225)(SEQ ID NO: 2144). FIG. 6B: A single nucleotide substitution at C170T ofthe H2.16 tumor cDNA is the only sequence change relative to thepublished L3 ribosomal allele. This substitution results in a T541 aminoacid substitution in the protein (SEQ ID NO: 2145).

FIGS. 7A and 7B. Identification of the Peptide Epitope Recognized by theTumor Specific CTL. FIG. 7A: CML assay to identify the peptiderecognized by tumor specific CTL. Target cells were labeled with ⁵¹Cr(15). During the ⁵¹Cr incubation samples of B/C.N cells were incubatedwith 1 μM peptide L3₄₈₋₅₆(I54), 100 μM L3₄₈₋₅₆(T54) or 100 μM peptideL3₄₅₋₅₄(I54). Target cells were incubated with the indicated ratios oftumor specific Cytotoxic T Lymphocytes for 4 hours at 37° C. andpercentage specific lysis was determined. This experiment was repeatedat least three times with similar results. FIG. 7B: Titration of peptideL3₄₈₋₅₆ (I54). Target cells were labeled with ⁵¹Cr. During the ⁵¹Crincubation samples of B/C.N cells were incubated either with no peptideaddition (D) or with the indicated concentrations (1 μM, 10 nM, 1 nM) ofL3₄₈₋₅₆(I54) (▪), BCA 39 cells were included as a positive control (▴).Target cells were incubated with the indicated ratios of Tumor SpecificCytotoxic T Lymphocytes for 4 hours at 37° C. and percentage specificlysis was determined. The experiment was repeated twice with similarresults.

FIGS. 8A to 8C. Analysis of L3 Expressed by Each Cell Line. FIG. 8A:Sau3AI map of published rpL3 and H2.16. Shown above is the Sau3AIrestriction map for the published ribosomal protein L3 gene (Top), andfor H2.16 (Bottom). Digestion of cDNA for the published L3 sequencegenerates fragments of 200, 355, 348, 289, and 84 bp. The pattern forH2.16 is identical except for an extra Sau3AI site at position 168caused by the C170T. This results in a 168 bp digestion product in placeof the 200 bp fragment. FIG. 8B: The BCA tumors express both L3 alleles.RT-PCR products generated from each cell line or from vH2.16 weregenerated using L3 specific primers and then digested with Sau3AI, andresolved on a 3% agarose gel for 2 hours at 80 volts. FIG. 8C: TheImmunogenic L3 allele is expressed at greatly reduced levels in B/C.N,BCB13, and Thymus. L3 specific RT-PCR products from each indicatedsample were generated using a ³²P end labeled 5 prime PCR primer. No PCRproduct was observed when RNA for each sample was used as template forPCR without cDNA synthesis, indicating that no sample was contaminatedwith genomic DNA. The PCR products were gel purified to ensure purity,digested with Sau3AI, and resolved on a 3% agarose gel for 15 hours at60 volts. No PCR product was observed in a control PCR sample that hadno template added to it. This result has been reproduced a total of 3times.

FIGS. 9A to 9C. Immunization with iL3 is Immunoprotective. FIG. 9A:Immunization with H2.16 induces tumor specific CTL. Balb/c mice(2/group) were immunized by subcutaneous injection with 5×10⁶ pfu ofvH2.16, or control vector v7.5/tk. Seven days later splenocytes wereharvested and restimulated with peptide L3₄₈₋₅₆(I54) (26). Five daysfollowing the second restimulation the lymphocytes were tested in achromium release assay as described in FIG. 11. The L3₄₈₋₅₆(I54) peptidewas used at a 1 micromolar concentration, and the L3₄₈₋₅₆(T54) peptidewas used at a 100 micromolar concentration. Similar results wereobtained when the immunization experiment was repeated. FIGS. 9B and 9C:Female Balb/cByJ mice were immunized as indicated (27). The mice werechallenged by SC injection with 200,000 viable BCA 34 tumor cells intothe abdominal wall. Data is from day 35 post challenge. These data arerepresentative of 4 independent experiments.

FIGS. 10A and 10B. FIG. 10A: C35 coding sequence with translation; (SEQID NO: 2)5′ and 3′ untranslated regions are shown in lowercase letters(SEQ ID NO: 2146). The predicted prenylation site, CVIL, at the 3′terminus is boxed. The asterisk (“*”) represents a termination codon andsignifies the end of the protein sequence. FIG. 10B: Genomic alignmentof C35 gene on chromosome 17.

FIGS. 11A and 11B. C35 Expression in Breast Carcinoma. C35 was labeledwith ³²P in a random priming reaction and hybridized to Northern blotsat 10⁶ cpm/ml. Each blot was stripped and re-probed with GAPDH orBeta-actin to normalize mRNA loads. The numbers indicate densitometryratios normalized against GAPDH/Beta-actin. A value of 1 has beenassigned to normal cell line H16N2, and all values are relative to thelevel of expression in the normal cell line. FIG. 11A: C35 expression inbreast epithelial cell lines. FIG. 11B: C35 expression in primary breasttissue/tumors. 300 ng mRNA was electrophoresed on 0.8% alkaline agarosegels, then blotted to GENESCREEN PLUS™, except leftmost panel of Bloaded with 1 μg mRNA from 3 primary tumors and 1 normal tissue control(Real Tumor Blots, Invitrogen). Similar exposures are shown for allblots.

FIG. 12. C35 Expression in Bladder Carcinoma. C35 was labeled with ³²Pin a random priming reaction and hybridized to a Northern blot of tumorand normal RNA at 10⁶ cpm/ml. The blot was stripped and re-probed withBeta-actin to normalize mRNA loads. The numbers indicate densitometryratios normalized against Beta-actin. Values are relative to the levelof expression in the normal bladder samples. 300 ng mRNA waselectrophoresed on 0.8% alkaline agarose gels, then blotted toGENESCREEN PLUS™.

FIGS. 13A and 13B. FACS Analysis with Anti-C35 Antibodies. FIG. 13A:Breast cell lines were stained with (top panel) sera from mice immunizedwith Line 1 cells infected with C35 recombinant retrovirus, and (bottompanel) 2C3 purified monoclonal antibody or isotype control. FIG. 13B:Bladder cell lines stained with 2C3 purified monoclonal antibody orisotype control.

FIGS. 14A and 14B. Inhibition of Tumor Growth in Presence of 2C3Antibody. 21NT breast tumor cells (FIG. 14A) or H16N2 normal breastepithelial cells (FIG. 14B) were incubated with the indicatedconcentrations of 2C3 anti-C35 monoclonal antibody or a non-specificisotype control antibody. Cell growth was measured by XTT assayfollowing 72 hour incubation in the presence or absence of antibodies.

FIGS. 15A and 15B. CTL stimulated with C35 expressing dendritic cellsspecifically lyse C35+Breast (21NT) and Bladder (ppT11A3) tumor celllines, with minimal activity against normal breast (MEC), immortalizednon-tumorigenic breast (H16N2) and bladder (SV-HUC) cell lines, or an NKsensitive cell line (K562). FIG. 15A: T cell line 4 was generated fromnormal human PBL. FIG. 15B: T cell clone 10G3 was selected from line 4for C35-specific activity. Target cell lines MEC, ppT11A3 and SV-HUC arenaturally HLA-A2 positive. Target cell lines 21NT and H16N2 weretransected with HLA-A2 to provide a required MHC restriction element.

FIGS. 16A and 16B. Cytokine Release from T Cell Clone 10G3 uponStimulation with Targets. FIG. 16A: IFN-gamma secretion. FIG. 16B:TNF-alpha secretion. Breast and bladder target cell lines weredistinguished by the presence or absence of expression of HLA-A2 and C35tumor antigen, an amino terminal 50 amino acid fragment of C35(C35-50aa), or the irrelevant mouse L3 ribosomal protein. Each markerwas either endogenously expressed or introduced by transfection of anHLA-A2.1 construct (pSV2.A2), or by infection with a vacciniarecombinant of C35 (vv.C35, vv.C35-50aa), L3 (vv.L3), or HLA-A2 (vv.A2)

FIGS. 17A and 17B. Effect of anti-CD40 ligand antibody (anti-CD154) inblocking the reactivity of murine T cells to specific transplantationantigens. DBA/2 (H-2^(d)) mice were immunized with 10⁷ C57B1/6 (H-2^(b))spleen cells intraperitoneally and, in addition, were injected witheither saline or 0.5 mg monoclonal anti-CD40 ligand antibody (MR1,anti-CD154, Pharmingen 09021D) administered both at the time ofimmunization and two days later. On day 10 following immunization,spleen cells from these mice were removed and stimulated in vitro witheither C57B1/6 or control allogeneic C3H(H-2^(k)) spleen cells that hadbeen irradiated (20 Gy). After 5 days of in vitro stimulation, C57B1/6and C3H specific cytolytic responses were assayed at variouseffector:target ratios by ⁵¹Cr release assay from specific labeledtargets, in this case, either C3H or C57B1/6 dendritic cells pulsed withsyngeneic spleen cell lysates. Significant cytotoxicity was inducedagainst the control C3H alloantigens in both saline and anti-CD154treated mice (FIG. 17A) whereas a cytotoxic response to C57B1/6 wasinduced in the saline treated mice but not the anti-CD154 treated mice(FIG. 17B).

FIG. 18. GM-CSF Production by Line 4 After Stimulation with Native21NT-A2 Tumor, H16N2-A2 Pulsed with Different C35 Peptides, or H16N2-A2Infected with C35 Recombinant Vaccinia Virus. T cell line 4 wasgenerated by stimulating normal donor T cells for 12 days each withautologous dendritic cells (DC) and then autologous monocytes infectedwith C35 recombinant vaccinia.virus. Weekly stimulation was continuedwith allo PBL and the 21NT tumor transfected with HLA-A2/Kb (21NT-A2).For the experiment depicted here, the T cells were restimulated in vitroat 10⁶ T cells per well with 5×10⁴ irradiated (2500 rads) H16N2-A2/Kbpulsed with 1 ug/ml of C35 peptides 9-17, 77-85, 104-112, or 105-113 and10⁵ irradiated allo PBL per well with IL2 (20 U/ml) and IL-7 (10 ng/ml)in AIM-V/5% human AB serum. After two (2) rounds of stimulation for 7days, T cells were tested for induction of GM-CSF secretion followingincubation with different stimulators pulsed or not pulsed with 1 ug/mlof peptide or infected with vvC35 or vvWT at MOI=1. T cells (5000) wereincubated with 25000 of the various stimulator cells overnight inAIM-V/5% human AB serum in triplicate.

FIG. 19. C35-Specific ELISA of Hybridoma Supernatants. Results of arepresentative ELISA experiment involving hybridoma clones withdemonstrated specificity for C35.

FIG. 20. Western Blot with C35-Specific Antibodies. Western BlotImmunodetection was performed with supernatant from selected hybridomaclones. Antibodies from 4 hybridomas (1B3, 1F2, 3E10, 11B10) reactedspecifically with hC35 protein in this assay. Results for antibodies1B3, 1F2, and 3E10 are shown.

FIG. 21. Immunohistochemistry with 1F2 Antibody. Monoclonal antibody 1F2was shown to have utility for immunohistochemical staining of primarybreast tumor sections. This Figure demonstrates that monoclonal antibody1F2 can detect high levels of endogenous C35 expression in human breasttumors, with little or no staining of normal breast tissue.Specifically, this Figure shows strong staining of a section of invasivebreast adenocarcinoma from patient 01A6, while normal breast tissue fromthe same patient is negative.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from itsnative environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.

In the present invention, a “membrane” C35 protein is one expressed onthe cell surface through either direct or indirect association with thelipid bilayer, including, in particular, through prenylation of acarboxyl-terminal amino acid motif. Prenylation involves the covalentmodification of a protein by the addition of either a farnesyl orgeranylgeranyl isoprenoid. Prenylation occurs on a cysteine residuelocated near the carboxyl-terminus of a protein. The C35 polypeptidecontains the amino acids Cys-Val-Ile-Leu at positions 112-115 of SEQ IDNO:2, with the Leu being the C terminal residue of the polypeptide. Themotif Cys-X-X-Leu SEQ ID NO:2142, where “X” represents any aliphaticamino acid, results in the addition of a 20 carbon geranylgeranyl grouponto the Cys residue. Generally, following addition of this lipid thethree terminal amino acid residues are cleaved off the polypeptide, andthe lipid group is methylated. Prenylation promotes the membranelocalization of most proteins, with sequence motifs in the polypeptidebeing involved in directing the prenylated protein to the plasma,nuclear, or golgi membranes. Prenylation plays a role in protein-proteininteractions, and many prenylated proteins are involved in signaltransduction. Examples of prenylated proteins include Ras and thenuclear lamin B. (Zhang, F. L. and Casey, P. J., Ann. Rev. Biochem.65:241-269 (1996)). The C35 protein has been detected on the surface oftwo breast tumor cell lines by fluorescence analysis employing as aprimary reagent a mouse anti-human C35 antiserum (FIGS. 4A-4C).

In the present invention, a “secreted” C35 protein refers to a proteincapable of being directed to the ER, secretory vesicles, or theextracellular space as a result of a signal sequence, as well as a C35protein released into the extracellular space without necessarilycontaining a signal sequence. If the C35 secreted protein is releasedinto the extracellular space, the C35 secreted protein can undergoextracellular processing to produce a “mature” C35 protein. Release intothe extracellular space can occur by many mechanisms, includingexocytosis and proteolytic cleavage.

As used herein, a C35 “polynucleotide” refers to a molecule having anucleic acid sequence contained in SEQ ID NO:1. For example, the C35polynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without the signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a C35 “polypeptide”refers to a molecule having the translated amino acid sequence generatedfrom the polynucleotide as broadly defined.

In specific embodiments, the polynucleotides of the invention are lessthan 300 nt, 200 nt, 100 nt, 50 nt, 15 nt, 10 nt, or 7 nt in length. Ina further embodiment, polynucleotides of the invention comprise at least15 contiguous nucleotides of C35 coding sequence, but do not compriseall or a portion of any C35 intron. In another embodiment, the nucleicacid comprising C35 coding sequence does not contain coding sequences ofa genomic flanking gene (i.e., 5′ or 3′ to the C35 gene in the genome).

In the present invention, the full length C35 coding sequence isidentified as SEQ ID NO: 1.

A C35 “polynucleotide” also refers to isolated polynucleotides whichencode the C35 polypeptides, and polynucleotides closely relatedthereto.

A C35 “polynucleotide” also refers to isolated polynucleotides whichencode the amino acid sequence shown in SEQ ID NO: 2, or a biologicallyactive fragment thereof.

A C35 “polynucleotide” also includes those polynucleotides capable ofhybridizing, under stringent hybridization conditions, to sequencescontained in SEQ ID NO: 1, the complement thereof, or the cDNA withinthe deposited clone. “Stringent hybridization conditions” refers to anovernight incubation at 42° in a solution comprising 50% formamide,5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65°.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA), or to a complementarystretch of T (or U) residues, would not be included in the definition of“polynucleotide,” since such a polynucleotide would hybridize to anynucleic acid molecule containing a poly (A) stretch or the complementthereof (e.g., practically any double-stranded cDNA clone).

The C35 polynucleotide can be composed of any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. For example, C35 polynucleotides can be composed of single-and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the C35 polynucleotides can be composed of triple-strandedregions comprising RNA or DNA or both RNA and DNA. C35 polynucleotidesmay also contain one or more modified bases or DNA or RNA backbonesmodified for stability or for other reasons. “Modified” bases include,for example, tritylated bases and unusual bases such as inosine. Avariety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

C35 polypeptides can be composed of amino acids joined to each other bypeptide bonds or modified peptide bonds, i.e., peptide isosteres, andmay contain amino acids other than the 20 gene-encoded amino acids. TheC35 polypeptides may be modified by either natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theC35 polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. It will be appreciatedthat the same type of modification may be present in the same or varyingdegrees at several sites in a given C35 polypeptide. Also, a given C35polypeptide may contain many types of modifications. C35 polypeptidesmay be branched, for example, as a result of ubiquitination, and theymay be cyclic, with or without branching. Cyclic, branched, and branchedcyclic C35 polypeptides may result from posttranslation naturalprocesses or may be made by synthetic methods. Modifications includeacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. (See, for instance, Proteins—StructureAnd Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12(1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al.,Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO: 1” refers to a C35 polynucleotide sequence while “SEQ ID NO:2” refers to a C35 polypeptide sequence.

A C35 polypeptide “having biological activity” refers to polypeptidesexhibiting activity similar to, but not necessarily identical to, anactivity of a C35 polypeptide, including mature forms, as measured in aparticular biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof the C35 polypeptide, but rather substantially similar to thedose-dependence in a given activity as compared to the C35 polypeptide(i.e., the candidate polypeptide will exhibit greater activity or notmore than about 25-fold less and, preferably, not more than abouttenfold less activity, and most preferably, not more than aboutthree-fold less activity relative to the C35 polypeptide.)

C35 Polynucleotides and Polypeptides

A 348 base pair fragment of C35 was initially isolated by subtractivehybridization of poly-A RNA from tumor and normal mammary epithelialcell lines derived from the same patient with primary and infiltratingintraductal mammary carcinoma. Band, V. et al., Cancer Res. 50:7351-7357(1990). Employing primers based on this sequence and that of anoverlapping EST sequence (Accession No. W57569), a cDNA that includesthe full-length C35 coding sequence was then amplified and cloned fromthe BT-20 breast tumor cell line (ATCC, HTB-19). This C35 cDNA containsthe entire coding region identified as SEQ ID NO: 1. The C35 cloneincludes, in addition to the 348 bp coding sequence, 167 bp of 3′untranslated region. The open reading frame begins at an N-terminalmethionine located at nucleotide position 1, and ends at a stop codon atnucleotide position 348 (FIG. 1A). A representative clone containing allor most of the sequence for SEQ ID NO:1 was deposited with the AmericanType Culture Collection (“ATCC”) on Aug. 1, 2000, and was given the ATCCDeposit Number PTA-2310. The ATCC is located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was madepursuant to the terms of the Budapest Treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure.

Therefore, SEQ ID NO: 1 and the translated SEQ ID NO: 2 are sufficientlyaccurate and otherwise suitable for a variety of uses well known in theart and described further below. For instance, SEQ ID NO: 1 is usefulfor designing nucleic acid hybridization probes that will detect nucleicacid sequences contained in SEQ ID NO: 1 or the cDNA contained in thedeposited clone. These probes will also hybridize to nucleic acidmolecules in biological samples, thereby enabling a variety of forensicand diagnostic methods of the invention. Similarly, polypeptidesidentified from SEQ ID NO:2 may be used to generate antibodies whichbind specifically to C35, or to stimulate T cells which are specific forC35 derived peptide epitopes in association with MHC molecules on thecell surface.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO: 1 and the predicted translated amino acid sequence identified as SEQID NO:2. The nucleotide sequence of the deposited C35 clone can readilybe determined by sequencing the deposited clone in accordance with knownmethods. The predicted C35 amino acid sequence can then be verified fromsuch deposits. Moreover, the amino acid sequence of the protein encodedby the deposited clone can also be directly determined by peptidesequencing or by expressing the protein in a suitable host cellcontaining the deposited human C35 cDNA, collecting the protein, anddetermining its sequence.

The present invention also relates to the C35 gene corresponding to SEQID NO:1, or the deposited clone. The C35 gene can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include preparing probes or primers from thedisclosed sequence and identifying or amplifying the C35 gene fromappropriate sources of genomic material.

Also provided in the present invention are species homologs of C35.Species homologs may be isolated and identified by making suitableprobes or primers from the sequences provided herein and screening asuitable nucleic acid source for the desired homologue.

By “C35 polypeptide(s)” is meant all forms of C35 proteins andpolypeptides described herein. The C35 polypeptides can be prepared inany suitable manner. Such polypeptides include isolated naturallyoccurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. Means for preparing such polypeptides arewell understood in the art.

The C35 polypeptides may be in the form of the membrane protein or asecreted protein, including the mature form, or may be a part of alarger protein, such as a fusion protein (see below). It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, prosequences, sequences which aid inpurification, such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

C35 polypeptides are preferably provided in an isolated form, andpreferably are substantially purified. A recombinantly produced versionof a C35 polypeptide, including the secreted polypeptide, can besubstantially purified by the one step method described in Smith andJohnson, Gene 67:3140 (1988). C35 polypeptides also can be purified fromnatural or recombinant sources using antibodies of the invention raisedagainst the C35 protein in methods which are well known in the art.

In one embodiment, the present invention is directed to an isolatedpolypeptide capable of eliciting a cytotoxic T lymphocyte and/or helperT lymphocyte response in a human subject, the isolated polypeptidecomprising, or, alternatively, consisting of, one or more C35 peptideepitopes or C35 peptide epitope analogs. In a preferred embodiment, saidone or more C35 peptide epitopes are selected from the group consistingof: amino acids E4 to P12 of SEQ ID NO:2, amino acids S9 to V17 of SEQID NO:2, amino acids S21 to Y29 of SEQ ID NO:2, G22 to C30 of SEQ IDNO:2, amino acids I25 to C33 of SEQ ID NO:2, amino acids T38 to V46 ofSEQ ID NO:2, amino acids G61 to I69 of SEQ ID NO:2, amino acids T62 toN70 of SEQ ID NO:2, amino acids G63 to G71 of SEQ ID NO:2, amino acidsF65 to L73 of SEQ ID NO:2, amino acids I67 to F75 of SEQ ID NO:2, aminoacids K77 to Y85 of SEQ ID NO:2, amino acids Q72 to E86 of SEQ ID NO:2,amino acids G81 to L89 of SEQ ID NO:2, amino acids K104 to C112 of SEQID NO:2, amino acids K104 to V113 of SEQ ID NO:2, amino acids I105 toV113 of SEQ ID NO:2, and amino acids N107 to L115 of SEQ ID NO:2. In apreferred embodiment, the isolated polypeptides comprising one or moreC35 peptide epitopes (e.g., one or more octamers, nonamers, decamers,15mers, or 20mers in Tables 1-3 or 5-6) or C35 peptide epitope analogs(e.g., an analog listed in Table 4) are not more than 114 amino acids inlength, more preferably not more than 110 amino acids in length, morepreferably not more than 105 amino acids in length, more preferably notmore than 100 amino acids in length, more preferably not more than 95amino acids in length, more preferably not more than 90 amino acids inlength, more preferably not more than 85 amino acids in length, morepreferably not more than 80 amino acids in length, more preferably notmore than 75 amino acids in length, more preferably not more than 70amino acids in length, more preferably not more than 65 amino acids inlength, more preferably not more than 60 amino acids in length, morepreferably not more than 55 amino acids in length, more preferably notmore than 50 amino acids in length, more preferably not more than 45amino acids in length, more preferably not more than 40 amino acids inlength, more preferably not more than 35 amino acids in length, morepreferably not more than 30 amino acids in length, more preferably notmore than 25 amino acids in length, more preferably 20 amino acids inlength, more preferably 15 amino acids in length, more preferably 14,13, 12, 11, 10, 9 or 8 amino acids in length. Of course, although notexplicitly listed here, isolated polypeptides of any length between, forexample, 8 and 100 amino acids, comprising C35 peptide epitopes or C35peptide epitope analogs are likewise contemplated by the presentinvention. In a preferred embodiment, the isolated polypeptide is afragment of the C35 polypeptide shown in SEQ ID NO:2 and FIG. 1B. Inanother embodiment, the present invention is directed to an isolatedpolypeptide capable of eliciting a cytotoxic T lymphocyte and/or helperT lymphocyte response in a human subject, the isolated polypeptidecomprising, or, alternatively, consisting of multiple C35 peptideepitopes. In a particularly preferred embodiment, saidmulti-epitopepolypeptide is selected from the group consisting of: aminoacids T101 to V113 of SEQ ID NO:2, amino acids E100 to V113 of SEQ IDNO:2, amino acids G99 to V113 of SEQ ID NO:2, amino acids I93 to V113 ofSEQ ID NO:2, amino acids D88 to V113 of SEQ ID NO:2, amino acids P84 toV113 of SEQ ID NO:2, amino acids K77 to V113 of SEQ ID NO:2, amino acidsQ72 to V113 of SEQ ID NO:2, amino acids F65 to V113 of SEQ ID NO:2, andamino acids L59 to V113 of SEQ ID NO:2. In another preferred embodiment,the present invention is directed to a fusion protein comprising atleast one C35 peptide epitope listed in Tables 1-3 or 5-6, or a C35peptide epitope analog listed in Table 4. In one embodiment, the atleast one C35 peptide epitope or C35 peptide epitope analog is fused toa heterologous (i.e., non-C35) polypeptide. In another preferredembodiment, said fusion protein comprises two or more C35 peptideepitopes or two or more C35 peptide epitope analogs, either as ahomopolymer or a heteropolymer. In another preferred embodiment, thefusion proteins of the present invention comprise at least one C35peptide epitope analog joined to at least one C35 peptide epitope. In afurther embodiment, the epitopes/analogs are joined by an amino acidspacer or linker.

The present invention is further directed to a pharmaceuticalcomposition for use as a vaccine comprising such isolated polypeptidesand fusion proteins.

The present invention is further directed to a method for stimulating acytotoxic T lymphocyte and/or a helper T lymphocyte response in a humanpatient comprising administering to said patient an immunogenicallyeffective amount of the pharmaceutical composition of the invention.

Polynucleotide and Polypeptide Variants

“Variant” refers to a polynucleotide or polypeptide differing from theC35 polynucleotide or polypeptide, but retaining essential propertiesthereof. Generally, variants are overall closely similar, and, in manyregions, identical to the C35 polynucleotide or polypeptide.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the C35polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence shown of SEQ ID NO:1, the ORF (open reading frame),or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to anucleotide sequence or polypeptide sequence of the presence inventioncan be determined conventionally using known computer programs. Apreferred method for determining the best overall match between a querysequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe FASTDB computer program based on the algorithm of Brutlag et al.,Comp. App. Biosci. 6:237245 (1990). In a sequence alignment the queryand subject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, ktuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or to the amino acid sequence encodedby deposited DNA clone, can be determined conventionally using knowncomputer programs. A preferred method for determining the best overallmatch between a query sequence (a sequence of the present invention) anda subject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al., Comp. App. Biosci. 6:237245 (1990). In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, ktuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the Nterminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the Nterminus. The 10 unpaired residues represent 10% of thesequence (number of residues at the N- and C-termini not matched/totalnumber of residues in the query sequence) so 10% is subtracted from thepercent identity score calculated by the FASTDB program. If theremaining 90 residues were perfectly matched the final percent identitywould be 90%. In another example, a 90 residue subject sequence iscompared with a 100 residue query sequence. This time the deletions areinternal deletions so there are no residues at the N- and C-termini ofthe subject sequence which are not matched/aligned with the query. Inthis case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only residue positions outside the N- andC-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to be made forthe purposes of the present invention.

The C35 variants may contain alterations in the coding regions,noncoding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which 510, 15, or 12 amino acids are substituted,deleted, or added in any combination are also preferred. C35polynucleotide variants can be produced for a variety of reasons, e.g.,to optimize codon expression for a particular host (change codons in thehuman mRNA to those preferred by a bacterial host such as E. coli).

Naturally occurring C35 variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985).) Also, allelic variants can occur as“tandem alleles” which are highly homologous sequences that occur atdifferent loci on chromosomes of an organism. These allelic variants canvary at either the polynucleotide and/or polypeptide level.Alternatively, nonnaturally occurring variants may be produced bymutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the C35 polypeptides. For instance, one or more aminoacids can be deleted from the Nterminus or Cterminus of the secretedprotein without substantial loss of biological function. The authors ofRon et al., J. Biol. Chem. 268: 29842988 (1993), reported variant KGFproteins having heparin binding activity even after deleting 3, 8, or 27aminoterminal amino acid residues. Similarly, Interferon gamma exhibitedup to ten times higher activity after deleting 810 amino acid residuesfrom the carboxy terminus of this protein (Dobeli et al., J.Biotechnology 7:199216 (1988)).

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem 268:2210522111 (1993))conducted extensive mutational analysis of human cytokine IL1a. Theyused random mutagenesis to generate over 3,500 individual IL1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwildtype.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes C35 polypeptide variants which showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as to have little effect on activity.For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., Science247:13061310 (1990), wherein the authors indicate that there are twomain strategies for studying the tolerance of an amino acid sequence tochange.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:10811085 (1989).) The resulting mutant molecules can then betested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the smallsized amino acids Ala, Ser, Thr, Met,and Gly.

Besides conservative amino acid substitution, variants of C35 include(i) substitutions with one or more of the nonconserved amino acidresidues, where the substituted amino acid residues may or may not beone encoded by the genetic code, or (ii) substitution with one or moreof amino acid residues having a substituent group, or (iii) fusion ofthe mature polypeptide with another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), or (iv) fusion of the polypeptide withadditional amino acids, such as an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

For example, C35 polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331340 (1967); Robbinset al., Diabetes 36: 838845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307377 (1993).)

Polynucleotide and Polypeptide Fragments

In the present invention, a “polynucleotide fragment” refers to a shortpolynucleotide having a nucleic acid sequence contained in the depositedclone or shown in SEQ ID NO:1. The short nucleotide fragments arepreferably at least about 15 nt, and more preferably at least about 20nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length. A fragment “at least 20 ntin length,” for example, is intended to include 20 or more contiguousbases from the cDNA sequence contained in the deposited clone or thenucleotide sequence shown in SEQ ID NO:1. These nucleotide fragments areuseful as diagnostic probes and primers as discussed herein. Of course,larger fragments (e.g., at least 50, 100, 150, 200, 250, 300nucleotides) are preferred.

Moreover, representative examples of C35 polynucleotide fragmentsinclude, for example, fragments having a sequence from about nucleotidenumber 150, 51100, 101150, 151200, 201250, 251300, or 301 to the end ofSEQ ID NO:1 or the cDNA contained in the deposited clone. In thiscontext “about” includes the particularly recited ranges, larger orsmaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus orat both termini. Preferably, these fragments encode a polypeptide whichhas biological activity. More preferably, these polynucleotides can beused as probes or primers as discussed herein.

In the present invention, a “polypeptide fragment” refers to a shortamino acid sequence contained in SEQ ID NO:2 and FIG. 1B or encoded bythe cDNA contained in the deposited clone. Protein fragments may be“freestanding,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number120, 2140, 4160, 6180, 81100, or 101 to the end of the coding region.Moreover, polypeptide fragments can comprise about 7, 8, 9, 15, 20, 30,40, 50, 60, 70, 80, 90, or 100 amino acids in length. In this context“about” includes the particularly recited ranges or lengths, larger orsmaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme orat both extremes.

Preferred polypeptide fragments include the secreted C35 protein as wellas the mature form. Further preferred polypeptide fragments include thesecreted C35 protein or the mature form having a continuous series ofdeleted residues from the amino or the carboxy terminus, or both.Further preferred polypeptide fragments include fragments of the C35polypeptide comprising one or more C35 peptide epitopes.

As mentioned above, even if deletion of one or more amino acids from theNterminus of a protein results in modification or loss of one or morebiological functions of the protein, other biological activities maystill be retained. Thus, the ability of shortened C35 muteins to induceand/or bind to antibodies which recognize the complete or mature formsof the polypeptides generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the Nterminus. Whether a particular polypeptide lackingNterminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a C35 muteinwith a large number of deleted Nterminal amino acid residues may retainsome biological or immunogenic activities. In fact, in the case of C35peptide epitopes, peptides composed of as few as 9, 8, or even 7 C35amino acid residues often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the C35 aminoacid sequence shown in SEQ ID NO:2, up to the Threonine residue atposition number 105 and polynucleotides encoding such polypeptides.

Also as mentioned above, even if deletion of one or more amino acidsfrom the Cterminus of a protein results in modification or loss of oneor more biological functions of the protein, other biological activitiesmay still be retained. Thus, the ability of the shortened C35 mutein toinduce cytotoxic T lymphocytes (CTLs) and/or helper T lymphocytes (HTLs)and/or to bind to antibodies which recognize the complete or matureforms of the polypeptide generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the Cterminus. Whether a particular polypeptide lackingCterminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a C35 muteinwith a large number of deleted Cterminal amino acid residues may retainsome biological or immunogenic activities.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the C35 polypeptide shown in SEQ ID NO:2, up to the valineresidue at position number 10, and polynucleotides encoding suchpolypeptides

Moreover, the invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini. Inpreferred embodiments, the invention is directed to peptides havingresidues: E4 to P12, S9 to V17; V10 to V17; E16 to V23; E16 to R24; E16to I25; S21 to Y29; S21 to F35; G22 to C30; I25 to C33; C30 to T38; E31to Y39; E36 to A43; A37 to A45; A37 to V46; T38 to V46; Y39 to V46; S44to I53; A45 to I53; G52 to L59; E54 to T62; S57 to F75; R58 to I67; L59to V113; G61 to I69; T62 to N70, G63 to G71, G63 to F83; F65 to L73; F65to V113; E66 to L73; E66 to V74; I67 to F75; K77 to Y85; K77 to V113;Q72 to E86; Q72 to V113; G81 to L89; F83 to E103; P84 to V113; D88 toA96; D88 to V113; L89 to A96; A92 to T101; I93 to V113; R95 to L102; A96to K104; G99 to V113, E100 to V113, T101 to V113; K104 to C112; K104 toV113; I105 to V113; I105 to I114; or N107 to L115 of SEQ ID NO:2 andpolynucleotides encoding such polypeptides.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases.

The human EST sequences referred to below were identified in a BLASTsearch of the EST database. These sequences are believed to be partialsequences of the cDNA inserts identified in the recited GenBankaccession numbers. No homologous sequences were identified in a searchof the annotated GenBank database. The Expect value (E) is a parameterthat describes the number of hits one can “expect” to see just by chancewhen searching a database of a particular size. It decreasesexponentially with the Score (S) that is assigned to a match between twosequences. Essentially, the E value describes the random backgroundnoise that exists for matches between sequences. In BLAST 2.0, theExpect value is also used instead of the P value (probability) to reportthe significance of matches. For example, an E value of 1 assigned to ahit can be interpreted as meaning that in a database of the current sizeone might expect to see 1 match with a similar score simply by chance.

For example, the following sequences are related to SEQ ID NO: 1,GenBank Accession Nos.: AA971857 (SEQ ID NO:3); W57569 (SEQ ID NO:4);AI288765 (SEQ ID NO:5); W65390 (SEQ ID NO:6); W37432 (SEQ ID NO: 7);N42748 (SEQ ID NO:8); AA971638 (SEQ ID NO:9); R22331 (SEQ ID NO:10);AA308370 (SEQ ID NO:11); AA285089 (SEQ ID NO:12); R68901 (SEQ ID NO:13);AA037285 (SEQ ID NO:14); H94832 (SEQ ID NO:15); H96058 (SEQ ID NO:16);H56522 (SEQ ID NO:17); AA935328 (SEQ ID NO: 18); AW327450 (SEQ IDNO:19); AW406075 (SEQ ID NO:20); AW406223 (SEQ ID NO:21); AI909652 (SEQID NO:22); AA026773 (SEQ ID NO: 23); H96055 (SEQ ID NO:24); H12836 (SEQID NO:25); R22401 (SEQ ID NO:26); N34596 (SEQ ID NO:27); W32121 (SEQ IDNO:28); T84927 (SEQ ID NO:29); R63575 (SEQ ID NO:30); R23139 (SEQ IDNO:31); AA337071 (SEQ ID NO:32); AA813244 (SEQ ID NO:33); AA313422 (SEQID NO:34); N31910 (SEQ ID NO:35); N42693 (SEQ ID NO:36); N32532 (SEQ IDNO:37); AA375119 (SEQ ID NO:38); R32153 (SEQ ID NO:39); R23369 (SEQ IDNO:40); AA393628 (SEQ ID NO:41); H12779 (SEQ ID NO:42); AI083674 (SEQ IDNO:43); AA284919 (SEQ ID NO:44); AA375286 (SEQ ID NO:45); AA830592 (SEQID NO:46); H95363 (SEQ ID NO:47); T92052 (SEQ ID NO:48); AI336555 (SEQID NO:49); AI285284 (SEQ ID NO:50); AA568537 (SEQ ID NO:51); AI041967(SEQ ID NO:52); W44577 (SEQ ID NO:53); R22332 (SEQ ID NO:54); N27088(SEQ ID NO:55); H96418 (SEQ ID NO:56); AI025384 (SEQ ID NO:57); AA707623(SEQ ID NO:58); AI051009 (SEQ ID NO:59); AA026774 (SEQ ID NO:60); W51792(SEQ ID NO:61); AI362693 (SEQ ID NO:62); AA911823 (SEQ ID NO:63); H96422(SEQ ID NO:64); AI800991 (SEQ ID NO:65); AI525314 (SEQ ID NO:66);AI934846 (SEQ ID NO:67); AI937133 (SEQ ID NO:68); AW006797 (SEQ IDNO:69); AI914716 (SEQ ID NO:70); AI672936 (SEQ ID NO:71); W61294 (SEQ IDNO:72); AI199227 (SEQ ID NO:73); AI499727 (SEQ ID NO:74); R32154 (SEQ IDNO:75); AI439771 (SEQ ID NO:76); AA872671 (SEQ ID NO:77); AA502178 (SEQID NO:78); N26715 (SEQ ID NO:79); AA704668 (SEQ ID NO:80); R68799 (SEQID NO:81); H56704 (SEQ ID NO:82); AI360416 (SEQ ID NO:83).

Thus, in one embodiment the present invention is directed topolynucleotides comprising the polynucleotide fragments and full-lengthpolynucleotide (e.g. the coding region) described herein exclusive ofone or more of the above-recited ESTs. Also, the nucleotide sequences inSEQ ID NO: 152, SEQ ID NO: 154, and SEQ ID NO: 156 are excluded from thepresent invention.

Also preferred are C35 polypeptide and polynucleotide fragmentscharacterized by structural or functional domains. Preferred embodimentsof the invention include fragments that comprise MHC binding epitopesand prenylation sites.

Other preferred fragments are biologically active C35 fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the C35 polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

Epitopes & Antibodies

Cellular peptides derived by degradation of endogenously synthesizedproteins are translocated into a pre-Golgi compartment where they bindto Class I or Class II MHC molecules for transport to the cell surface.These class I MHC:peptide complexes are the target antigens for specificCD8+ cytotoxic T cells. Since all endogenous proteins “turn over,”peptides derived from any cytoplasmic or nuclear protein may bind to anMHC molecule and be transported for presentation at the cell surface.This allows T cells to survey a much larger representation of cellularproteins than antibodies which are restricted to recognizeconformational determinants of only those proteins that are eithersecreted or integrated at the cell membrane. The T cell receptor antigenbinding site interacts with determinants of both the peptide and thesurrounding MHC. T cell specificity must, therefore, be defined in termsof an MHC:peptide complex. The specificity of peptide binding to MHCmolecules is very broad and of relatively low affinity in comparison tothe antigen binding site of specific antibodies. Class I-bound peptidesare generally 8-10 residues in length that accommodate amino acid sidechains of restricted diversity at certain key positions that matchpockets in the MHC peptide binding site. These key features of peptidesthat bind to a particular MHC molecule constitute a peptide bindingmotif.

The term “derived” when used to discuss a peptide epitope is a synonymfor “prepared.” A derived epitope can be isolated from a natural source,or it can be synthesized in accordance with standard protocols in theart. Synthetic epitopes can comprise artificial amino acids “amino acidmimetics,” such as D isomers of natural occurring L amino acids ornon-natural amino acids such as cyclohexylalanine. A derived/preparedepitope can be an analog of a native epitope.

An “epitope” is the collective features of a molecule, such as primary,secondary and tertiary peptide structure, and charge, that together forma site recognized by an immunoglobulin, T cell receptor or HLA molecule.Alternatively, an epitope can be defined as a set of amino acid residueswhich is involved in recognition by a particular immunoglobulin, or inthe context of T cells, those residues necessary for recognition by Tcell receptor proteins and/or Major Histocompatibility Complex (MHC)receptors. Epitopes are present in nature, and can be isolated, purifiedor otherwise prepared/derived by humans. For example, epitopes can beprepared by isolation from a natural source, or they can be synthesizedin accordance with standard protocols in the art. Synthetic epitopes cancomprise artificial amino acids “amino acid mimetics,” such as D isomersof natural occurring L amino acids or non-natural amino acids such ascyclohexylalanine. Throughout this disclosure, the terms epitope andpeptide are often used interchangeably. Also, the term epitope as usedherein is generally understood to encompass analogs of said epitopes.

It is to be appreciated that protein or polypeptide molecules thatcomprise one or more C35 peptide epitopes of the invention as well asadditional amino acid(s) are still within the bounds of the invention.In certain embodiments, there is a limitation on the length of apolypeptide of the invention of, for example, not more than 114 aminoacids, not more than 110 amino acids, not more than 100 amino acids, notmore than 95 amino acids, not more than 90 amino acids, not more than 85amino acids, not more than 80 amino acids, not more than 75 amino acids,not more than 70 amino acids, not more than 65 amino acids, not morethan 60 amino acids, not more than 55 amino acids, not more than 50amino acids, not more than 45 amino acids, not more than 40 amino acids,not more than 35 amino acids, not more than 30 amino acids, not morethan 25 amino acids, 20 amino acids, 15 amino acids, or 14, 13, 12, 11,10, 9 or 8 amino acids. In some instances, the embodiment that islength-limited occurs when the protein/polypeptide comprising an epitopeof the invention comprises a region (i.e., a contiguous series of aminoacids) having 100% identity with a native sequence. In order to avoidthe definition of epitope from reading, e.g., on whole naturalmolecules, there is a limitation on the length of any region that has100% identity with a native polypeptide sequence. Thus, for apolypeptide comprising an epitope of the invention and a region with100% identity with the native C35 polypeptide sequence, the region with100% identity to the native sequence generally has a length of: lessthan or equal to 114 amino acids, more often less than or equal to 100amino acids, often less than or equal to 85 amino acids, often less thanor equal to 75 amino acids, often less than or equal to 65 amino acids,and often less than or equal to 50 amino acids. In certain embodiments,the C35 polypeptide of the invention comprises a peptide having a regionwith less than 50 amino acids that has 100% identity to a native peptidesequence, in any increment of amino acids (i.e., 49, 48, 47, 46, 45, 44,43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26,25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5) down to 5 amino acids. Preferably, such C35 polypeptide comprisesone or more C35 peptide epitopes.

Accordingly, polypeptide or protein sequences longer than 100 aminoacids are within the scope of the invention, so long as they do notcomprise any contiguous sequence of more than 114 amino acids that have100% identity with a native polypeptide sequence. For any polypeptidethat has five contiguous residues or less that correspond to a nativesequence, there is no limitation on the maximal length of thatpolypeptide in order to fall within the scope of the invention. In oneembodiment, the polypeptide of the invention comprising one or more C35peptide epitopes is less than 60 residues long in any increment down toeight amino acid residues.

An “immunogenic peptide” or “peptide epitope” is a peptide that willbind an HLA molecule and induce a cytotoxic T lymphocyte (CTL) responseand/or a helper T lymphocyte (HTL) response. Thus, immunogenic peptidesof the invention are capable of binding to an appropriate HLA moleculeand thereafter inducing a cytotoxic T lymphocyte (CTL) response, or ahelper T lymphocyte (HTL) response, to the peptide.

The term “motif” refers to a pattern of residues in an amino acidsequence of defined length, usually a peptide of from about 8 to about13 amino acids for a class I HLA motif and from about 16 to about 25amino acids for a class II HLA motif, which is recognized by aparticular HLA molecule. Motifs are typically different for each HLAprotein encoded by a given human HLA allele. These motifs often differin their pattern of the primary and secondary anchor residues.

A “protective immune response” or “therapeutic immune response” refersto a cytotoxic T lymphocyte (CTL) and/or an helper T lymphocyte (HTL)response to an antigen derived from an pathogenic antigen (e.g., anantigen from an infectious agent or a tumor antigen), which in some wayprevents or at least partially arrests disease symptoms, side effects orprogression. The immune response may also include an antibody responsewhich has been facilitated by the stimulation of helper T cells.

The term “residue” refers to an amino acid or amino acid mimeticincorporated into a peptide or protein by an amide bond or amide bondmimetic.

“Synthetic peptide” refers to a peptide that is not naturally occurring,but is man-made using such methods as chemical synthesis or recombinantDNA technology.

As used herein, a “vaccine” is a composition that contains one or morepeptide epitopes of the invention, see, e.g., Tables 1-3 and 5-6,exclusive of peptide E-100 to R-109, and a pharmaceutically acceptablecarrier. There are numerous embodiments of vaccines in accordance withthe invention, such as by a cocktail of one or more peptides; apolyepitopic peptide comprising one or more peptides of the invention;or nucleic acids that encode such peptides or polypeptides, e.g., aminigene that encodes a polyepitopic peptide. The “one or more peptides”or “one or more epitopes” can include, for example, at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more peptides or epitopesof the invention. The peptides or polypeptides can optionally bemodified, such as by lipidation, addition of targeting or othersequences. HLA class I-binding peptides of the invention can be linkedto HLA class II-binding peptides, to facilitate activation of bothcytotoxic T lymphocytes and helper T lymphocytes. Vaccines can comprisepeptide pulsed antigen presenting cells, e.g., dendritic cells.

In a preferred embodiment, the isolated polypeptides of the presentinvention comprise or, alternatively, consist of one or more of thefollowing C35 peptide epitopes: amino acids E4 to P12 of SEQ ID NO:2,amino acids S9 to V17 of SEQ ID NO:2, amino acids S21 to Y29 of SEQ IDNO:2, G22 to C30 of SEQ ID NO: 2, amino acids I25 to C33 of SEQ ID NO:2,amino acids T38 to V46 of SEQ ID NO:2, amino acids G61 to I69 of SEQ IDNO:2, amino acids T62 to N70 of SEQ ID NO:2, amino acids G63 to G71 ofSEQ ID NO:2, amino acids F65 to L73 of SEQ ID NO:2, amino acids I67 toF75 of SEQ ID NO:2, amino acids K77 to Y85 of SEQ ID NO:2, amino acidsQ72 to E86 of SEQ ID NO:2, amino acids G81 to L89 of SEQ ID NO:2, aminoacids K104 to C112 of SEQ ID NO:2, amino acids K104 to V113 of SEQ IDNO:2, amino acids I105 to V113 of SEQ ID NO:2, or amino acids N107 toL115 of SEQ ID NO:2. In another embodiment, said polypeptides comprisingor, alternatively, consisting of one or more C35 peptide epitopes areselected from the group consisting of: T101 to V113 of SEQ ID NO:2, G99to V113 of SEQ ID NO:2, E100 to V113 of SEQ ID NO:2, I93 to V113 of SEQID NO:2, D88 to V113 of SEQ ID NO:2, P84 to V113 of SEQ ID NO:2, K77 toV113 of SEQ ID NO:2, Q72 to V113 of SEQ ID NO:2, F65 to V113 of SEQ IDNO:2, and L59 to V113 of SEQ ID NO:2. It is contemplated that fragmentsof C35 peptide epitopes and polypeptides comprising fragments of C35peptide epitopes of the invention will, in some instances, also beuseful for stimulating a cytotoxic T lymphocyte response. Thus, thepresent invention includes fragments of the C35 peptide epitopes inwhich 1, 2, 3, 4, 5 or more amino acids of the peptide sequence providedhave been deleted from either the amino terminus or the carboxy terminusof the peptide. In addition, it is contemplated that larger fragments ofthe C35 polypeptide that contain one or more of the peptide epitopes ofthe invention may also be used to stimulate a CTL response in a patient.It is further contemplated that polypeptides that comprise one or morepeptide epitopes of the present invention in addition to heterologous,i.e., non-C35, flanking sequences may also be used to stimulate a CTLresponse.

In addition to the specific C35 peptide epitopes specifically listedabove, many other peptide epitopes are contemplated by the presentinvention. Thus, the isolated polypeptides of the present inventioncomprising or, alternatively, consisting of, one or more C35 peptideepitopes include the following 8mers (residues correspond to SEQ ID NO:2and FIG. 1B): M1 to T8; S2 to S9; G3 to V10; E4 to A11; P5 to P12; G6 toP13; Q7 to P14; T8 to E15; S9 to E16; V10 to V17; A11 to E18; P12 toP19; P13 to G20; P14 to S21; E15 to G22; E16 to V23; V17 to R24; E18 toI25; P19 to V26; G20 to V27; S21 to E28; G22 to Y29; V23 to C30; R24 toE31; I25 to P32; V26 to C33; V27 to G34; E28 to F35; Y29 to E36; C30 toA37; E31 to T38; P32 to Y39; C33 to L40; G34 to E41; F35 to L42; E36 toA43; A37 to S44; T38 to A45; Y39 to V46; L40 to K47; E41 to E48; L42 toQ49; A43 to Y50; S44 to P51; A45 to G52; V46 to I53; K47 to E54; E48 toI55; Q49 to E56; Y50 to S57; P51 to R58; G52 to L59; I53 to G60; E54 toG61; I55 to T62; E56 to G63; S57 to A64; R58 to F65; L59 to E66; G60 toI67; G61 to E68; T62 to I69; G63 to N70; A64 to G71; F65 to Q72; E66 toL73; I67 to V74; E68 to F75; I69 to S76; N70 to K77; G71 to L78; Q72 toE79; L73 to N80; V74 to G81; F75 to G82; S76 to F83; K77 to P84; L78 toY85; E79 to E86; N80 to K87; G81 to D88; G82 to L89; F83 to I90; P84 toE91; Y85 to A92; E86 to I93; K87 to R94; D88 to R95; L89 to A96; I90 toS97; E91 to N98; A92 to G99; I93 to E100; R94 to T101; R95 to L102; A96to E103; S97 to K104; N98 to I105; G99 to T106; E100 to N107; T101 toS108; L102 to R109; E103 to P110; K104 to P111; I105 to C112; T106 toV113; N107 to I114; and S108 to L115.

In a further embodiment, the isolated polypeptides of the presentinvention comprising or, alternatively, consisting of, one or more C35peptide epitopes include the following 9mers (residues correspond to SEQID NO:2 and FIG. 1B): M1 to S9; S2 to V10; G3 to A11; E4 to P12; P5 toP13; G6 to P14; Q7 to E15; T8 to E16; S9 to V17; V10 to E18; A11 to P19;P12 to G20; P13 to S21; P14 to G22; E15 to V23; E16 to R24; V17 to I25;E18 to V26; P19 to V27; G20 to E28; S21 to Y29; G22 to C30; V23 to E31;R24 to P32; I25 to C33; V26 to G34; V27 to F35; E28 to E36; Y29 to A37;C30 to T38; E31 to Y39; P32 to L40; C33 to E41; G34 to L42; F35 to A43;E36 to S44; A37 to A45; T38 to V46; Y39 to K47; L40 to E48; E41 to Q49;L42 to Y50; A43 to P51; S44 to G52; A45 to I53; V46 to E54; K47 to I55;E48 to E56; Q49 to S57; Y50 to R58; P51 to L59; G52 to G60; I53 to G61;E54 to T62; I55 to G63; E56 to A64; S57 to F65; R58 to E66; L59 to I67;G60 to E68; G61 to I69; T62 to N70; G63 to G71; A64 to Q72; F65 to L73;E66 to V74; I67 to F75; E68 to S76; I69 to K77; N70 to L78; G71 to E79;Q72 to N80; L73 to G81; V74 to G82; F75 to F83; S76 to P84; K77 to Y85;L78 to E86; E79 to K87; N80 to D88; G81 to L89; G82 to I90; F83 to E91;P84 to A92; Y85 to I93; E86 to R94; K87 to R95; D88 to A96; L89 to S97;I90 to N98; E91 to G99; A92 to E100; I93 to T101; R94 to L102; R95 toE103; A96 to K104; S97 to I105; N98 to T106; G99 to N107; E100 to S108;T101 to R109; L102 to P110; E103 to P111; K104 to C112; I105 to V113;T106 to I114; and N107 to L115.

In a further embodiment, the isolated polypeptides of the presentinvention comprising or, alternatively, consisting of, one or more C35peptide epitopes include the following 10mers (residues correspond toSEQ ID NO:2 and FIG. 1B): M1 to V10; S2 to A11; G3 to P12; E4 to P13; P5to P14; G6 to E15; Q7 to E16; T8 to V17; S9 to E18; V10 to P19; A11 toG20; P12 to S21; P13 to G22; P14 to V23; E15 to R24; E16 to I25; V17 toV26; E18 to V27; P19 to E28; G20 to Y29; S21 to C30; G22 to E31; V23 toP32; R24 to C33; I25 to G34; V26 to F35; V27 to E36; E28 to A37; Y29 toT38; C30 to Y39; E31 to L40; P32 to E41; C33 to L42; G34 to A43; F35 toS44; E36 to A45; A37 to V46; T38 to K47; Y39 to E48; L40 to Q49; E41 toY50; L42 to P51; A43 to G52; S44 to I53; A45 to E54; V46 to I55; K47 toE56; E48 to S57; Q49 to R58; Y50 to L59; P51 to G60; G52 to G61; I53 toT62; E54 to G63; I55 to A64; E56 to F65; S57 to E66; R58 to I67; L59 toE68; G60 to I69; G61 to N70; T62 to G71; G63 to Q72; A64 to L73; F65 toV74; E66 to F75; I67 to S76; E68 to K77; I69 to L78; N70 to E79; G71 toN80; Q72 to G81; L73 to G82; V74 to F83; F75 to P84; S76 to Y85; K77 toE86; L78 to K87; E79 to D88; N80 to L89; G81 to I90; G82 to E91; F83 toA92; P84 to I93; Y85 to R94; E86 to R95; K87 to A96; D88 to S97; L89 toN98; I90 to G99; E91 to E100; A92 to T101; I93 to L102; R94 to E103; R95to K104; A96 to I105; S97 to T106; N98 to N107; G99 to S108; E100 toR109; T101 to P110; L102 to P111; E103 to C112; K104 to V113; I105 toI114; T106 to L115.

In a further embodiment, the isolated polypeptides of the presentinvention comprising or, alternatively, consisting of, one or more C35peptide epitopes include the following 11mers (residues correspond toSEQ ID NO:2 and FIG. 1B): M1 to A11; S2 to P12; G3 to P13; E4 to P14; P5to E15; G6 to E16; Q7 to V17; T8 to E18; S9 to P19; V10 to G20; A11 toS21; P12 to G22; P13 to V23; P14 to R24; E15 to I25; E16 to V26; V17 toV27; E18 to E28; P19 to Y29; G20 to C30; S21 to E31; G22 to P32; V23 toC33; R24 to G34; I25 to F35; V26 to E36; V27 to A37; E28 to T38; Y29 toY39; C30 to L40; E31 to E41; P32 to L42; C33 to A43; G34 to S44; F35 toA45; E36 to V46; A37 to K47; T38 to E48; Y39 to Q49; L40 to Y50; E41 toP51; L42 to G52; A43 to I53; S44 to E54; A45 to I55; V46 to E56; K47 toS57; E48 to R58; Q49 to L59; Y50 to G60; P51 to G61; G52 to T62; I53 toG63; E54 to A64; I55 to F65; E56 to E66; S57 to I67; R58 to E68; L59 toI69; G60 to N70; G61 to G71; T62 to Q72; G63 to L73; A64 to V74; F65 toF75; E66 to S76; I67 to K77; E68 to L78; I69 to E79; N70 to N80; G71 toG81; Q72 to G82; L73 to F83; V74 to P84; F75 to Y85; S76 to E86; K77 toK87; L78 to D88; E79 to L89; N80 to I90; G81 to E91; G82 to A92; F83 toI93; P84 to R94; Y85 to R95; E86 to A96; K87 to S97; D88 to N98; L89 toG99; I90 to E100; E91 to T101; A92 to L102; I93 to E103; R94 to K104;R95 to I105; A96 to T106; S97 to N107; N98 to S108; G99 to R109; E100 toP110; T101 to P111; L102 to C112; E103 to V113; K104 to I114; I105 toL115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, C35peptide epitopes include the following 12mers (residues correspond toSEQ ID NO:2 and FIG. 1B): M1 to P12; S2 to P13; G3 to P14; E4 to E15; P5to E16; G6 to V17; Q7 to E18; T8 to P19; S9 to G20; V10 to S21; A11 toG22; P12 to V23; P13 to R24; P14 to I25; E15 to V26; E16 to V27; V17 toE28; E18 to Y29; P19 to C30; G20 to E31; S21 to P32; G22 to C33; V23 toG34; R24 to F35; I25 to E36; V26 to A37; V27 to T38; E28 to Y39; Y29 toL40; C30 to E41; E31 to L42; P32 to A43; C33 to S44; G34 to A45; F35 toV46; E36 to K47; A37 to E48; T38 to Q49; Y39 to Y50; L40 to P51; E41 toG52; L42 to I53; A43 to E54; S44 to I55; A45 to E56; V46 to S57; K47 toR58; E48 to L59; Q49 to G60; Y50 to G61; P51 to T62; G52 to G63; I53 toA64; E54 to F65; I55 to E66; E56 to I67; S57 to E68; R58 to I69; L59 toN70; G60 to G71; G61 to Q72; T62 to L73; G63 to V74; A64 to F75; F65 toS76; E66 to K77; I67 to L78; E68 to E79; I69 to N80; N70 to G81; G71 toG82; Q72 to F83; L73 to P84; V74 to Y85; F75 to E86; S76 to K87; K77 toD88; L78 to L89; E79 to I90; N80 to E91; G81 to A92; G82 to I93; F83 toR94; P84 to R95; Y85 to A96; E86 to S97; K87 to N98; D88 to G99; L89 toE100; I90 to T101; E91 to L102; A92 to E103; I93 to K104; R94 to I105;R95 to T106; A96 to N107; S97 to S108; N98 to R109; G99 to P110; E100 toP111; T101 to C112; L102 to V113; E103 to I114; K104 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 13mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to P13; S2 to P14; G3 to E15;E4 to E16; P5 to V17; G6 to E18; Q7 to P19; T8 to G20; S9 to S21; V10 toG22; A11 to V23; P12 to R24; P13 to I25; P14 to V26; E15 to V27; E16 toE28; V17 to Y29; E18 to C30; P19 to E31; G20 to P32; S21 to C33; G22 toG34; V23 to F35; R24 to E36; I25 to A37; V26 to T38; V27 to Y39; E28 toL40; Y29 to E41; C30 to L42; E31 to A43; P32 to S44; C33 to A45; G34 toV46; F35 to K47; E36 to E48; A37 to Q49; T38 to Y50; Y39 to P51; L40 toG52; E41 to I53; L42 to E54; A43 to I55; S44 to E56; A45 to S57; V46 toR58; K47 to L59; E48 to G60; Q49 to G61; Y50 to T62; P51 to G63; G52 toA64; I53 to F65; E54 to E66; I55 to I67; E56 to E68; S57 to I69; R58 toN70; L59 to G71; G60 to Q72; G61 to L73; T62 to V74; G63 to F75; A64 toS76; F65 to K77; E66 to L78; I67 to E79; E68 to N80; I69 to G81; N70 toG82; G71 to F83; Q72 to P84; L73 to Y85; V74 to E86; F75 to K87; S76 toD88; K77 to L89; L78 to I90; E79 to E91; N80 to A92; G81 to I93; G82 toR94; F83 to R95; P84 to A96; Y85 to S97; E86 to N98; K87 to G99; D88 toE100; L89 to T101; I90 to L102; E91 to E103; A92 to K104; I93 to I105;R94 to T106; R95 to N107; A96 to S108; S97 to R109; N98 to P110; G99 toP111; E100 to C112; T101 to V113; L102 to I114; E103 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 14mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to P14; S2 to E15; G3 to E16;E4 to V17; P5 to E18; G6 to P19; Q7 to G20; T8 to S21; S9 to G22; V10 toV23; A11 to R24; P12 to I25; P13 to V26; P14 to V27; E15 to E28; E16 toY29; V17 to C30; E18 to E31; P19 to P32; G20 to C33; S21 to G34; G22 toF35; V23 to E36; R24 to A37; I25 to T38; V26 to Y39; V27 to L40; E28 toE41; Y29 to L42; C30 to A43; E31 to S44; P32 to A45; C33 to V46; G34 toK47; F35 to E48; E36 to Q49; A37 to Y50; T38 to P51; Y39 to G52; L40 toI53; E41 to E54; L42 to I55; A43 to E56; S44 to S57; A45 to R58; V46 toL59; K47 to G60; E48 to G61; Q49 to T62; Y50 to G63; P51 to A64; G52 toF65; I53 to E66; E54 to I67; I55 to E68; E56 to I69; S57 to N70; R58 toG71; L59 to Q72; G60 to L73; G61 to V74; T62 to F75; G63 to S76; A64 toK77; F65 to L78; E66 to E79; I67 to N80; E68 to G81; I69 to G82; N70 toF83; G71 to P84; Q72 to Y85; L73 to E86; V74 to K87; F75 to D88; S76 toL89; K77 to I90; L78 to E91; E79 to A92; N80 to I93; G81 to R94; G82 toR95; F83 to A96; P84 to S97; Y85 to N98; E86 to G99; K87 to E100; D88 toT101; L89 to L102; I90 to E103; E91 to K104; A92 to I105; I93 to T106;R94 to N107; R95 to S108; A96 to R109; S97 to P110; N98 to P111; G99 toC112; E100 to V113; T101 to I114; L102 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 15mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to E15; S2 to E16; G3 to V17;E4 to E18; P5 to P19; G6 to G20; Q7 to S21; T8 to G22; S9 to V23; V10 toR24; A11 to I25; P12 to V26; P13 to V27; P14 to E28; E15 to Y29; E16 toC30; V17 to E31; E18 to P32; P19 to C33; G20 to G34; S21 to F35; G22 toE36; V23 to A37; R24 to T38; I25 to Y39; V26 to L40; V27 to E41; E28 toL42; Y29 to A43; C30 to S44; E31 to A45; P32 to V46; C33 to K47; G34 toE48; F35 to Q49; E36 to Y50; A37 to P51; T38 to G52; Y39 to I53; L40 toE54; E41 to I55; L42 to E56; A43 to S57; S44 to R58; A45 to L59; V46 toG60; K47 to G61; E48 to T62; Q49 to G63; Y50 to A64; P51 to F65; G52 toE66; I53 to I67; E54 to E68; I55 to I69; E56 to N70; S57 to G71; R58 toQ72; L59 to L73; G60 to V74; G61 to F75; T62 to S76; G63 to K77; A64 toL78; F65 to E79; E66 to N80; I67 to G81; E68 to G82; I69 to F83; N70 toP84; G71 to Y85; Q72 to E86; L73 to K87; V74 to D88; F75 to L89; S76 toI90; K77 to E91; L78 to A92; E79 to I93; N80 to R94; G81 to R95; G82 toA96; F83 to S97; P84 to N98; Y85 to G99; E86 to E100; K87 to T101; D88to L102; L89 to E103; I90 to K104; E91 to I105; A92 to T106; I93 toN107; R94 to S108; R95 to R109; A96 to P110; S97 to P111; N98 to C112;G99 to V113; E100 to I114; T101 to L115.

In a further preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 16mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to E16; S2 to V17; G3 to E18;E4 to P19; P5 to G20; G6 to S21; Q7 to G22; T8 to V23; S9 to R24; V10 toI25; A11 to V26; P12 to V27; P13 to E28; P14 to Y29; E15 to C30; E16 toE31; V17 to P32; E18 to C33; P19 to G34; G20 to F35; S21 to E36; G22 toA37; V23 to T38; R24 to Y39; I25 to L40; V26 to E41; V27 to L42; E28 toA43; Y29 to S44; C30 to A45; E31 to V46; P32 to K47; C33 to E48; G34 toQ49; F35 to Y50; E36 to P51; A37 to G52; T38 to I53; Y39 to E54; L40 toI55; E41 to E56; L42 to S57; A43 to R58; S44 to L59; A45 to G60; V46 toG61; K47 to T62; E48 to G63; Q49 to A64; Y50 to F65; P51 to E66; G52 toI67; I53 to E68; E54 to I69; I55 to N70; E56 to G71; S57 to Q72; R58 toL73; L59 to V74; G60 to F75; G61 to S76; T62 to K77; G63 to L78; A64 toE79; F65 to N80; E66 to G81; I67 to G82; E68 to F83; I69 to P84; N70 toY85; G71 to E86; Q72 to K87; L73 to D88; V74 to L89; F75 to I90; S76 toE91; K77 to A92; L78 to I93; E79 to R94; N80 to R95; G81 to A96; G82 toS97; F83 to N98; P84 to G99; Y85 to E100; E86 to T101; K87 to L102; D88to E103; L89 to K104; I90 to I105; E91 to T106; A92 to N107; I93 toS108; R94 to R109; R95 to P110; A96 to P111; S97 to C112; N98 to V113;G99 to I114; E100 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 17mers: M1 to V17; S2 toE18; G3 to P19; E4 to G20; P5 to S21; G6 to G22; Q7 to V23; T8 to R24;S9 to I25; V10 to V26; A11 to V27; P12 to E28; P13 to Y29; P14 to C30;E15 to E31; E16 to P32; V17 to C33; E18 to G34; P19 to F35; G20 to E36;S21 to A37; G22 to T38; V23 to Y39; R24 to L40; I25 to E41; V26 to L42;V27 to A43; E28 to S44; Y29 to A45; C30 to V46; E31 to K47; P32 to E48;C33 to Q49; G34 to Y50; F35 to P51; E36 to G52; A37 to I53; T38 to E54;Y39 to I55; L40 to E56; E41 to S57; L42 to R58; A43 to L59; S44 to G60;A45 to G61; V46 to T62; K47 to G63; E48 to A64; Q49 to F65; Y50 to E66;P51 to I67; G52 to E68; I53 to I69; E54 to N70; I55 to G71; E56 to Q72;S57 to L73; R58 to V74; L59 to F75; G60 to S76; G61 to K77; T62 to L78;G63 to E79; A64 to N80; F65 to G81; E66 to G82; I67 to F83; E68 to P84;I69 to Y85; N70 to E86; G71 to K87; Q72 to D88; L73 to L89; V74 to I90;F75 to E91; S76 to A92; K77 to I93; L78 to R94; E79 to R95; N80 to A96;G81 to S97; G82 to N98; F83 to G99; P84 to E100; Y85 to T101; E86 toL102; K87 to E103; D88 to K104; L89 to I105; I90 to T106; E91 to N107;A92 to S108; I93 to R109; R94 to P110; R95 to P111; A96 to C112; S97 toV113; N98 to I114; G99 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 18mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to E18; S2 to P19; G3 to G20;E4 to S21; P5 to G22; G6 to V23; Q7 to R24; T8 to I25; S9 to V26; V10 toV27; A11 to E28; P12 to Y29; P13 to C30; P14 to E31; E15 to P32; E16 toC33; V17 to G34; E18 to F35; P19 to E36; G20 to A37; S21 to T38; G22 toY39; V23 to L40; R24 to E41; I25 to L42; V26 to A43; V27 to S44; E28 toA45; Y29 to V46; C30 to K47; E31 to E48; P32 to Q49; C33 to Y50; G34 toP51; F35 to G52; E36 to I53; A37 to E54; T38 to I55; Y39 to E56; L40 toS57; E41 to R58; L42 to L59; A43 to G60; S44 to G61; A45 to T62; V46 toG63; K47 to A64; E48 to F65; Q49 to E66; Y50 to I67; P51 to E68; G52 toI69; I53 to N70; E54 to G71; I55 to Q72; E56 to L73; S57 to V74; R58 toF75; L59 to S76; G60 to K77; G61 to L78; T62 to E79; G63 to N80; A64 toG81; F65 to G82; E66 to F83; I67 to P84; E68 to Y85; I69 to E86; N70 toK87; G71 to D88; Q72 to L89; L73 to I90; V74 to E91; F75 to A92; S76 toI93; K77 to R94; L78 to R95; E79 to A96; N80 to S97; G81 to N98; G82 toG99; F83 to E100; P84 to T101; Y85 to L102; E86 to E103; K87 to K104;D88 to I105; L89 to T106; I90 to N107; E91 to S108; A92 to R109; I93 toP110; R94 to P111; R95 to C112; A96 to V113; S97 to I114; N98 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 19mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to P19; S2 to G20; G3 to S21;E4 to G22; P5 to V23; G6 to R24; Q7 to I25; T8 to V26; S9 to V27; V10 toE28; A11 to Y29; P12 to C30; P13 to E31; P14 to P32; E15 to C33; E16 toG34; V17 to F35; E18 to E36; P19 to A37; G20 to T38; S21 to Y39; G22 toL40; V23 to E41; R24 to L42; I25 to A43; V26 to S44; V27 to A45; E28 toV46; Y29 to K47; C30 to E48; E31 to Q49; P32 to Y50; C33 to P51; G34 toG52; F35 to I53; E36 to E54; A37 to I55; T38 to E56; Y39 to S57; L40 toR58; E41 to L59; L42 to G60; A43 to G61; S44 to T62; A45 to G63; V46 toA64; K47 to F65; E48 to E66; Q49 to I67; Y50 to E68; P51 to I69; G52 toN70; I53 to G71; E54 to Q72; I55 to L73; E56 to V74; S57 to F75; R58 toS76; L59 to K77; G60 to L78; G61 to E79; T62 to N80; G63 to G81; A64 toG82; F65 to F83; E66 to P84; I67 to Y85; E68 to E86; I69 to K87; N70 toD88; G71 to L89; Q72 to I90; L73 to E91; V74 to A92; F75 to I93; S76 toR94; K77 to R95; L78 to A96; E79 to S97; N80 to N98; G81 to G99; G82 toE100; F83 to T101; P84 to L102; Y85 to E103; E86 to K104; K87 to I105;D88 to T106; L89 to N107; I90 to S108; E91 to R109; A92 to P110; I93 toP111; R94 to C112; R95 to V113; A96 to I114; S97 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 20mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to G20; S2 to S21; G3 to G22;E4 to V23; P5 to R24; G6 to I25; Q7 to V26; T8 to V27; S9 to E28; V10 toY29; A11 to C30; P12 to E31; P13 to P32; P14 to C33; E15 to G34; E16 toF35; V17 to E36; E18 to A37; P19 to T38; G20 to Y39; S21 to L40; G22 toE41; V23 to L42; R24 to A43; I25 to S44; V26 to A45; V27 to V46; E28 toK47; Y29 to E48; C30 to Q49; E31 to Y50; P32 to P51; C33 to G52; G34 toI53; F35 to E54; E36 to I55; A37 to E56; T38 to S57; Y39 to R58; L40 toL59; E41 to G60; L42 to G61; A43 to T62; S44 to G63; A45 to A64; V46 toF65; K47 to E66; E48 to I67; Q49 to E68; Y50 to I69; P51 to N70; G52 toG71; I53 to Q72; E54 to L73; I55 to V74; E56 to F75; S57 to S76; R58 toK77; L59 to L78; G60 to E79; G61 to N80; T62 to G81; G63 to G82; A64 toF83; F65 to P84; E66 to Y85; I67 to E86; E68 to K87; I69 to D88; N70 toL89; G71 to I90; Q72 to E91; L73 to A92; V74 to I93; F75 to R94; S76 toR95; K77 to A96; L78 to S97; E79 to N98; N80 to G99; G81 to E100; G82 toT101; F83 to L102; P84 to E103; Y85 to K104; E86 to I105; K87 to T106;D88 to N107; L89 to S108; I90 to R109; E91 to P110; A92 to P111; I93 toC112; R94 to V113; R95 to I114; A96 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 21mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to S21; S2 to G22; G3 to V23;E4 to R24; P5 to I25; G6 to V26; Q7 to V27; T8 to E28; S9 to Y29; V10 toC30; A11 to E31; P12 to P32; P13 to C33; P14 to G34; E15 to F35; E16 toE36; V17 to A37; E18 to T38; P19 to Y39; G20 to L40; S21 to E41; G22 toL42; V23 to A43; R24 to S44; I25 to A45; V26 to V46; V27 to K47; E28 toE48; Y29 to Q49; C30 to Y50; E31 to P51; P32 to G52; C33 to I53; G34 toE54; F35 to I55; E36 to E56; A37 to S57; T38 to R58; Y39 to L59; L40 toG60; E41 to G61; L42 to T62; A43 to G63; S44 to A64; A45 to F65; V46 toE66; K47 to I67; E48 to E68; Q49 to I69; Y50 to N70; P51 to G71; G52 toQ72; I53 to L73; E54 to V74; I55 to F75; E56 to S76; S57 to K77; R58 toL78; L59 to E79; G60 to N80; G61 to G81; T62 to G82; G63 to F83; A64 toP84; F65 to Y85; E66 to E86; I67 to K87; E68 to D88; I69 to L89; N70 toI90; G71 to E91; Q72 to A92; L73 to I93; V74 to R94; F75 to R95; S76 toA96; K77 to S97; L78 to N98; E79 to G99; N80 to E100; G81 to T101; G82to L102; F83 to E103; P84 to K104; Y85 to I105; E86 to T106; K87 toN107; D88 to S108; L89 to R109; I90 to P110; E91 to P111; A92 to C112;I93 to V113; R94 to I114; R95 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 22mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to G22; S2 to V23; G3 to R24;E4 to I25; P5 to V26; G6 to V27; Q7 to E28; T8 to Y29; S9 to C30; V10 toE31; A11 to P32; P12 to C33; P13 to G34; P14 to F35; E15 to E36; E16 toA37; V17 to T38; E18 to Y39; P19 to L40; G20 to E41; S21 to L42; G22 toA43; V23 to S44; R24 to A45; I25 to V46; V26 to K47; V27 to E48; E28 toQ49; Y29 to Y50; C30 to P51; E31 to G52; P32 to I53; C33 to E54; G34 toI55; F35 to E56; E36 to S57; A37 to R58; T38 to L59; Y39 to G60; L40 toG61; E41 to T62; L42 to G63; A43 to A64; S44 to F65; A45 to E66; V46 toI67; K47 to E68; E48 to I69; Q49 to N70; Y50 to G71; P51 to Q72; G52 toL73; I53 to V74; E54 to F75; I55 to S76; E56 to K77; S57 to L78; R58 toE79; L59 to N80; G60 to G81; G61 to G82; T62 to F83; G63 to P84; A64 toY85; F65 to E86; E66 to K87; I67 to D88; E68 to L89; I69 to I90; N70 toE91; G71 to A92; Q72 to I93; L73 to R94; V74 to R95; F75 to A96; S76 toS97; K77 to N98; L78 to G99; E79 to E100; N80 to T101; G81 to L102; G82to E103; F83 to K104; P84 to I105; Y85 to T106; E86 to N107; K87 toS108; D88 to R109; L89 to P110; I90 to P111; E91 to C112; A92 to V113;I93 to I114; R94 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 23mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to V23; S2 to R24; G3 to I25;E4 to V26; P5 to V27; G6 to E28; Q7 to Y29; T8 to C30; S9 to E31; V10 toP32; A11 to C33; P12 to G34; P13 to F35; P14 to E36; E15 to A37; E16 toT38; V17 to Y39; E18 to L40; P19 to E41; G20 to L42; S21 to A43; G22 toS44; V23 to A45; R24 to V46; I25 to K47; V26 to E48; V27 to Q49; E28 toY50; Y29 to P51; C30 to G52; E31 to I53; P32 to E54; C33 to I55; G34 toE56; F35 to S57; E36 to R58; A37 to L59; T38 to G60; Y39 to G61; L40 toT62; E41 to G63; L42 to A64; A43 to F65; S44 to E66; A45 to I67; V46 toE68; K47 to I69; E48 to N70; Q49 to G71; Y50 to Q72; P51 to L73; G52 toV74; I53 to F75; E54 to S76; I55 to K77; E56 to L78; S57 to E79; R58 toN80; L59 to G81; G60 to G82; G61 to F83; T62 to P84; G63 to Y85; A64 toE86; F65 to K87; E66 to D88; I67 to L89; E68 to I90; I69 to E91; N70 toA92; G71 to I93; Q72 to R94; L73 to R95; V74 to A96; F75 to S97; S76 toN98; K77 to G99; L78 to E100; E79 to T101; N80 to L102; G81 to E103; G82to K104; F83 to I105; P84 to T106; Y85 to N107; E86 to S108; K87 toR109; D88 to P110; L89 to P111; I90 to C112; E91 to V113; A92 to I114;I93 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 24mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to R24; S2 to I25; G3 to V26;E4 to V27; P5 to E28; G6 to Y29; Q7 to C30; T8 to E31; S9 to P32; V10 toC33; A11 to G34; P12 to F35; P13 to E36; P14 to A37; E15 to T38; E16 toY39; V17 to L40; E18 to E41; P19 to L42; G20 to A43; S21 to S44; G22 toA45; V23 to V46; R24 to K47; I25 to E48; V26 to Q49; V27 to Y50; E28 toP51; Y29 to G52; C30 to I53; E31 to E54; P32 to I55; C33 to E56; G34 toS57; F35 to R58; E36 to L59; A37 to G60; T38 to G61; Y39 to T62; L40 toG63; E41 to A64; L42 to F65; A43 to E66; S44 to I67; A45 to E68; V46 toI69; K47 to N70; E48 to G71; Q49 to Q72; Y50 to L73; P51 to V74; G52 toF75; I53 to S76; E54 to K77; I55 to L78; E56 to E79; S57 to N80; R58 toG81; L59 to G82; G60 to F83; G61 to P84; T62 to Y85; G63 to E86; A64 toK87; F65 to D88; E66 to L89; I67 to I90; E68 to E91; I69 to A92; N70 toI93; G71 to R94; Q72 to R95; L73 to A96; V74 to S97; F75 to N98; S76 toG99; K77 to E100; L78 to T101; E79 to L102; N80 to E103; G81 to K104;G82 to I105; F83 to T106; P84 to N107; Y85 to S108; E86 to R109; K87 toP110; D88 to P111; L89 to C112; I90 to V113; E91 to I114; A92 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 25mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to I25; S2 to V26; G3 to V27;E4 to E28; P5 to Y29; G6 to C30; Q7 to E31; T8 to P32; S9 to C33; V10 toG34; A11 to F35; P12 to E36; P13 to A37; P14 to T38; E15 to Y39; E16 toL40; V17 to E41; E18 to L42; P19 to A43; G20 to S44; S21 to A45; G22 toV46; V23 to K47; R24 to E48; I25 to Q49; V26 to Y50; V27 to P51; E28 toG52; Y29 to I53; C30 to E54; E31 to I55; P32 to E56; C33 to S57; G34 toR58; F35 to L59; E36 to G60; A37 to G61; T38 to T62; Y39 to G63; L40 toA64; E41 to F65; L42 to E66; A43 to I67; S44 to E68; A45 to I69; V46 toN70; K47 to G71; E48 to Q72; Q49 to L73; Y50 to V74; P51 to F75; G52 toS76; I53 to K77; E54 to L78; I55 to E79; E56 to N80; S57 to G81; R58 toG82; L59 to F83; G60 to P84; G61 to Y85; T62 to E86; G63 to K87; A64 toD88; F65 to L89; E66 to I90; I67 to E91; E68 to A92; I69 to I93; N70 toR94; G71 to R95; Q72 to A96; L73 to S97; V74 to N98; F75 to G99; S76 toE100; K77 to T101; L78 to L102; E79 to E103; N80 to K104; G81 to I105;G82 to T106; F83 to N107; P84 to S108; Y85 to R109; E86 to P110; K87 toP111; D88 to C112; L89 to V113; I90 to I114; E91 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 26mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to V26; S2 to V27; G3 to E28;E4 to Y29; P5 to C30; G6 to E31; Q7 to P32; T8 to C33; S9 to G34; V10 toF35; A11 to E36; P12 to A37; P13 to T38; P14 to Y39; E15 to L40; E16 toE41; V17 to L42; E18 to A43; P19 to S44; G20 to A45; S21 to V46; G22 toK47; V23 to E48; R24 to Q49; I25 to Y50; V26 to P51; V27 to G52; E28 toI53; Y29 to E54; C30 to I55; E31 to E56; P32 to S57; C33 to R58; G34 toL59; F35 to G60; E36 to G61; A37 to T62; T38 to G63; Y39 to A64; L40 toF65; E41 to E66; L42 to I67; A43 to E68; S44 to I69; A45 to N70; V46 toG71; K47 to Q72; E48 to L73; Q49 to V74; Y50 to F75; P51 to S76; G52 toK77; I53 to L78; E54 to E79; I55 to N80; E56 to G81; S57 to G82; R58 toF83; L59 to P84; G60 to Y85; G61 to E86; T62 to K87; G63 to D88; A64 toL89; F65 to I90; E66 to E91; I67 to A92; E68 to I93; I69 to R94; N70 toR95; G71 to A96; Q72 to S97; L73 to N98; V74 to G99; F75 to E100; S76 toT101; K77 to L102; L78 to E103; E79 to K104; N80 to I105; G81 to T106;G82 to N107; F83 to S108; P84 to R109; Y85 to P110; E86 to P111; K87 toC112; D88 to V113; L89 to I114; I90 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 27mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to V27; S2 to E28; G3 to Y29;E4 to C30; P5 to E31; G6 to P32; Q7 to C33; T8 to G34; S9 to F35; V10 toE36; A11 to A37; P12 to T38; P13 to Y39; P14 to L40; E15 to E41; E16 toL42; V17 to A43; E18 to S44; P19 to A45; G20 to V46; S21 to K47; G22 toE48; V23 to Q49; R24 to Y50; I25 to P51; V26 to G52; V27 to I53; E28 toE54; Y29 to I55; C30 to E56; E31 to S57; P32 to R58; C33 to L59; G34 toG60; F35 to G61; E36 to T62; A37 to G63; T38 to A64; Y39 to F65; L40 toE66; E41 to I67; L42 to E68; A43 to I69; S44 to N70; A45 to G71; V46 toQ72; K47 to L73; E48 to V74; Q49 to F75; Y50 to S76; P51 to K77; G52 toL78; I53 to E79; E54 to N80; I55 to G81; E56 to G82; S57 to F83; R58 toP84; L59 to Y85; G60 to E86; G61 to K87; T62 to D88; G63 to L89; A64 toI90; F65 to E91; E66 to A92; I67 to I93; E68 to R94; I69 to R95; N70 toA96; G71 to S97; Q72 to N98; L73 to G99; V74 to E100; F75 to T101; S76to L102; K77 to E103; L78 to K104; E79 to I105; N80 to T106; G81 toN107; G82 to S108; F83 to R109; P84 to P110; Y85 to P111; E86 to C112;K87 to V113; D88 to I114; L89 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 28mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to E28; S2 to Y29; G3 to C30;E4 to E31; P5 to P32; G6 to C33; Q7 to G34; T8 to F35; S9 to E36; V10 toA37; A11 to T38; P12 to Y39; P13 to L40; P14 to E41; E15 to L42; E16 toA43; V17 to S44; E18 to A45; P19 to V46; G20 to K47; S21 to E48; G22 toQ49; V23 to Y50; R24 to P51; I25 to G52; V26 to I53; V27 to E54; E28 toI55; Y29 to E56; C30 to S57; E31 to R58; P32 to L59; C33 to G60; G34 toG61; F35 to T62; E36 to G63; A37 to A64; T38 to F65; Y39 to E66; L40 toI67; E41 to E68; L42 to I69; A43 to N70; S44 to G71; A45 to Q72; V46 toL73; K47 to V74; E48 to F75; Q49 to S76; Y50 to K77; P51 to L78; G52 toE79; I53 to N80; E54 to G81; I55 to G82; E56 to F83; S57 to P84; R58 toY85; L59 to E86; G60 to K87; G61 to D88; T62 to L89; G63 to I90; A64 toE91; F65 to A92; E66 to I93; I67 to R94; E68 to R95; I69 to A96; N70 toS97; G71 to N98; Q72 to G99; L73 to E100; V74 to T101; F75 to L102; S76to E103; K77 to K104; L78 to I105; E79 to T106; N80 to N107; G81 toS108; G82 to R109; F83 to P110; P84 to P111; Y85 to C112; E86 to V113;K87 to I114; D88 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 29mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to Y29; S2 to C30; G3 to E31;E4 to P32; P5 to C33; G6 to G34; Q7 to F35; T8 to E36; S9 to A37; V10 toT38; A11 to Y39; P12 to L40; P13 to E41; P14 to L42; E15 to A43; E16 toS44; V17 to A45; E18 to V46; P19 to K47; G20 to E48; S21 to Q49; G22 toY50; V23 to P51; R24 to G52; I25 to I53; V26 to E54; V27 to I55; E28 toE56; Y29 to S57; C30 to R58; E31 to L59; P32 to G60; C33 to G61; G34 toT62; F35 to G63; E36 to A64; A37 to F65; T38 to E66; Y39 to I67; L40 toE68; E41 to I69; L42 to N70; A43 to G71; S44 to Q72; A45 to L73; V46 toV74; K47 to F75; E48 to S76; Q49 to K77; Y50 to L78; P51 to E79; G52 toN80; I53 to G81; E54 to G82; I55 to F83; E56 to P84; S57 to Y85; R58 toE86; L59 to K87; G60 to D88; G61 to L89; T62 to I90; G63 to E91; A64 toA92; F65 to I93; E66 to R94; I67 to R95; E68 to A96; I69 to S97; N70 toN98; G71 to G99; Q72 to E100; L73 to T101; V74 to L102; F75 to E103; S76to K104; K77 to I105; L78 to T106; E79 to N107; N80 to S108; G81 toR109; G82 to P110; F83 to P111; P84 to C112; Y85 to V113; E86 to I114;K87 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 30mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to C30; S2 to E31; G3 to P32;E4 to C33; P5 to G34; G6 to F35; Q7 to E36; T8 to A37; S9 to T38; V10 toY39; A11 to L40; P12 to E41; P13 to L42; P14 to A43; E15 to S44; E16 toA45; V17 to V46; E18 to K47; P19 to E48; G20 to Q49; S21 to Y50; G22 toP51; V23 to G52; R24 to I53; I25 to E54; V26 to I55; V27 to E56; E28 toS57; Y29 to R58; C30 to L59; E31 to G60; P32 to G61; C33 to T62; G34 toG63; F35 to A64; E36 to F65; A37 to E66; T38 to I67; Y39 to E68; L40 toI69; E41 to N70; L42 to G71; A43 to Q72; S44 to L73; A45 to V74; V46 toF75; K47 to S76; E48 to K77; Q49 to L78; Y50 to E79; P51 to N80; G52 toG81; I53 to G82; E54 to F83; I55 to P84; E56 to Y85; S57 to E86; R58 toK87; L59 to D88; G60 to L89; G61 to I90; T62 to E91; G63 to A92; A64 toI93; F65 to R94; E66 to R95; I67 to A96; E68 to S97; I69 to N98; N70 toG99; G71 to E100; Q72 to T101; L73 to L102; V74 to E103; F75 to K104;S76 to I105; K77 to T106; L78 to N107; E79 to S108; N80 to R109; G81 toP110; G82 to P111; F83 to C112; P84 to V113; Y85 to I114; E86 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 31mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to E31; S2 to P32; G3 to C33;E4 to G34; P5 to F35; G6 to E36; Q7 to A37; T8 to T38; S9 to Y39; V10 toL40; A11 to E41; P12 to L42; P13 to A43; P14 to S44; E15 to A45; E16 toV46; V17 to K47; E18 to E48; P19 to Q49; G20 to Y50; S21 to P51; G22 toG52; V23 to I53; R24 to E54; I25 to I55; V26 to E56; V27 to S57; E28 toR58; Y29 to L59; C30 to G60; E31 to G61; P32 to T62; C33 to G63; G34 toA64; F35 to F65; E36 to E66; A37 to I67; T38 to E68; Y39 to I69; L40 toN70; E41 to G71; L42 to Q72; A43 to L73; S44 to V74; A45 to F75; V46 toS76; K47 to K77; E48 to L78; Q49 to E79; Y50 to N80; P51 to G81; G52 toG82; I53 to F83; E54 to P84; I55 to Y85; E56 to E86; S57 to K87; R58 toD88; L59 to L89; G60 to I90; G61 to E91; T62 to A92; G63 to I93; A64 toR94; F65 to R95; E66 to A96; I67 to S97; E68 to N98; I69 to G99; N70 toE100; G71 to T101; Q72 to L102; L73 to E103; V74 to K104; F75 to I105;S76 to T106; K77 to N107; L78 to S108; E79 to R109; N80 to P110; G81 toP111; G82 to C112; F83 to V113; P84 to I114 and Y85 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 32mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to P32; S2 to C33; G3 to G34;E4 to F35; P5 to E36; G6 to A37; Q7 to T38; T8 to Y39; S9 to L40; V10 toE41; A11 to L42; P12 to A43; P13 to S44; P14 to A45; E15 to V46; E16 toK47; V17 to E48; E18 to Q49; P19 to Y50; G20 to P51; S21 to G52; G22 toI53; V23 to E54; R24 to I55; I25 to E56; V26 to S57; V27 to R58; E28 toL59; Y29 to G60; C30 to G61; E31 to T62; P32 to G63; C33 to A64; G34 toF65; F35 to E66; E36 to I67; A37 to E68; T38 to I69; Y39 to N70; L40 toG71; E41 to Q72; L42 to L73; A43 to V74; S44 to F75; A45 to S76; V46 toK77; K47 to L78; E48 to E79; Q49 to N80; Y50 to G81; P51 to G82; G52 toF83; I53 to P84; E54 to Y85; I55 to E86; E56 to K87; S57 to D88; R58 toL89; L59 to I90; G60 to E91; G61 to A92; T62 to I93; G63 to R94; A64 toR95; F65 to A96; E66 to S97; I67 to N98; E68 to G99; I69 to E100; N70 toT101; G71 to L102; Q72 to E103; L73 to K104; V74 to I105; F75 to T106;S76 to N107; K77 to S108; L78 to R109; E79 to P110; N80 to P111; G81 toC112; G82 to V113; F83 to I114 and P84 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 33mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to C33; S2 to G34; G3 to F35;E4 to E36; P5 to A37; G6 to T38; Q7 to Y39; T8 to L40; S9 to E41; V10 toL42; A11 to A43; P12 to S44; P13 to A45; P14 to V46; E15 to K47; E16 toE48; V17 to Q49; E18 to Y50; P19 to P51; G20 to G52; S21 to I53; G22 toE54; V23 to I55; R24 to E56; I25 to S57; V26 to R58; V27 to L59; E28 toG60; Y29 to G61; C30 to T62; E31 to G63; P32 to A64; C33 to F65; G34 toE66; F35 to I67; E36 to E68; A37 to I69; T38 to N70; Y39 to G71; L40 toQ72; E41 to L73; L42 to V74; A43 to F75; S44 to S76; A45 to K77; V46 toL78; K47 to E79; E48 to N80; Q49 to G81; Y50 to G82; P51 to F83; G52 toP84; I53 to Y85; E54 to E86; I55 to K87; E56 to D88; S57 to L89; R58 toI90; L59 to E91; G60 to A92; G61 to I93; T62 to R94; G63 to R95; A64 toA96; F65 to S97; E66 to N98; I67 to G99; E68 to E100; I69 to T101; N70to L102; G71 to E103; Q72 to K104; L73 to I105; V74 to T106; F75 toN107; S76 to S108; K77 to R109; L78 to P110; E79 to P111; N80 to C112;G81 to V113; G82 to I114 and F83 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 34mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to G34; S2 to F35; G3 to E36;E4 to A37; P5 to T38; G6 to Y39; Q7 to L40; T8 to E41; S9 to L42; V10 toA43; A11 to S44; P12 to A45; P13 to V46; P14 to K47; E15 to E48; E16 toQ49; V17 to Y50; E18 to P51; P19 to G52; G20 to I53; S21 to E54; G22 toI55; V23 to E56; R24 to S57; I25 to R58; V26 to L59; V27 to G60; E28 toG61; Y29 to T62; C30 to G63; E31 to A64; P32 to F65; C33 to E66; G34 toI67; F35 to E68; E36 to I69; A37 to N70; T38 to G71; Y39 to Q72; L40 toL73; E41 to V74; L42 to F75; A43 to S76; S44 to K77; A45 to L78; V46 toE79; K47 to N80; E48 to G81; Q49 to G82; Y50 to F83; P51 to P84; G52 toY85; I53 to E86; E54 to K87; I55 to D88; E56 to L89; S57 to I90; R58 toE91; L59 to A92; G60 to I93; G61 to R94; T62 to R95; G63 to A96; A64 toS97; F65 to N98; E66 to G99; I67 to E100; E68 to T101; I69 to L102; N70to E103; G71 to K104; Q72 to I105; L73 to T106; V74 to N107; F75 toS108; S76 to R109; K77 to P110; L78 to P111; E79 to C112; N80 to V113;G81 to I114 and G82 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 35mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to F35; S2 to E36; G3 to A37;E4 to T38; P5 to Y39; G6 to L40; Q7 to E41; T8 to L42; S9 to A43; V10 toS44; A11 to A45; P12 to V46; P13 to K47; P14 to E48; E15 to Q49; E16 toY50; V17 to P51; E18 to G52; P19 to I53; G20 to E54; S21 to I55; G22 toE56; V23 to S57; R24 to R58; I25 to L59; V26 to G60; V27 to G61; E28 toT62; Y29 to G63; C30 to A64; E31 to F65; P32 to E66; C33 to I67; G34 toE68; F35 to I69; E36 to N70; A37 to G71; T38 to Q72; Y39 to L73; L40 toV74; E41 to F75; L42 to S76; A43 to K77; S44 to L78; A45 to E79; V46 toN80; K47 to G81; E48 to G82; Q49 to F83; Y50 to P84; P51 to Y85; G52 toE86; I53 to K87; E54 to D88; I55 to L89; E56 to I90; S57 to E91; R58 toA92; L59 to I93; G60 to R94; G61 to R95; T62 to A96; G63 to S97; A64 toN98; F65 to G99; E66 to E100; I67 to T101; E68 to L102; I69 to E103; N70to K104; G71 to I105; Q72 to T106; L73 to N107; V74 to S108; F75 toR109; S76 to P110; K77 to P111; L78 to C112; E79 to V113; N80 to I114;G81 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 36mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E36; S2 to A37; G3 to T38; E4 to Y39; P5 to L40; G6 to E41; Q7 toL42; T8 to A43; S9 to S44; V10 to A45; A11 to V46; P12 to K47; P13 toE48; P14 to Q49; E15 to Y50; E16 to P51; V17 to G52; E18 to I53; P19 toE54; G20 to I55; S21 to E56; G22 to S57; V23 to R58; R24 to L59; I25 toG60; V26 to G61; V27 to T62; E28 to G63; Y29 to A64; C30 to F65; E31 toE66; P32 to I67; C33 to E68; G34 to I69; F35 to N70; E36 to G71; A37 toQ72; T38 to L73; Y39 to V74; L40 to F75; E41 to S76; L42 to K77; A43 toL78; S44 to E79; A45 to N80; V46 to G81; K47 to G82; E48 to F83; Q49 toP84; Y50 to Y85; P51 to E86; G52 to K87; I53 to D88; E54 to L89; I55 toI90; E56 to E91; S57 to A92; R58 to I93; L59 to R94; G60 to R95; G61 toA96; T62 to S97; G63 to N98; A64 to G99; F65 to E100; E66 to T101; I67to L102; E68 to E103; I69 to K104; N70 to I105; G71 to T106; Q72 toN107; L73 to S108; V74 to R109; F75 to P110; S76 to P111; K77 to C112;L78 to V113; E79 to I114; N80 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 37mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to A37; S2 to T38; G3 to Y39; E4 to L40; P5 to E41; G6 to L42; Q7 toA43; T8 to S44; S9 to A45; V10 to V46; A11 to K47; P12 to E48; P13 toQ49; P14 to Y50; E15 to P51; E16 to G52; V17 to I53; E18 to E54; P19 toI55; G20 to E56; S21 to S57; G22 to R58; V23 to L59; R24 to G60; I25 toG61; V26 to T62; V27 to G63; E28 to A64; Y29 to F65; C30 to E66; E31 toI67; P32 to E68; C33 to I69; G34 to N70; F35 to G71; A37 to L73; T38 toV74; Y39 to F75; L40 to S76; E41 to K77; L42 to L78; A43 to E79; S44 toN80; A45 to G81; V46 to G82; K47 to F83; E48 to P84; Q49 to Y85; Y50 toE86; P51 to K87; G52 to D88; I53 to L89; E54 to I90; I55 to E91; E56 toA92; S57 to I93; R58 to R94; L59 to R95; G60 to A96; G61 to S97; T62 toN98; G63 to G99; A64 to E100; F65 to T101; E66 to L102; I67 to E103; E68to K104; I69 to I105; N70 to T106; G71 to N107; Q72 to S108; L73 toR109; V74 to P110; F75 to P111; S76 to C112; K77 to V113; L78 to I114;E79 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 38mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to T38; S2 to Y39; G3 to L40; E4 to E41; P5 to L42; G6 to A43; Q7 toS44; T8 to A45; S9 to V46; V10 to K47; A11 to E48; P12 to Q49; P13 toY50; P14 to P51; E15 to G52; E16 to I53; V17 to E54; E18 to I55; P19 toE56; G20 to S57; S21 to R58; G22 to L59; V23 to G60; R24 to G61; I25 toT62; V26 to G63; V27 to A64; E28 to F65; Y29 to E66; C30 to I67; E31 toE68; P32 to I69; C33 to N70; G34 to G71; F35 to Q72; E36 to L73; A37 toV74; T38 to F75; Y39 to S76; L40 to K77; E41 to L78; L42 to E79; A43 toN80; S44 to G81; A45 to G82; V46 to F83; K47 to P84; E48 to Y85; Q49 toE86; Y50 to K87; P51 to D88; G52 to L89; I53 to I90; E54 to E91; I55 toA92; E56 to I93; S57 to R94; R58 to R95; L59 to A96; G60 to S97; G61 toN98; T62 to G99; G63 to E100; A64 to T101; F65 to L102; E66 to E103; I67to K104; E68 to I105; I69 to T106; N70 to N107; G71 to S108; Q72 toR109; L73 to P110; V74 to P111; F75 to C112; S76 to V113; K77 to I114;L78 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 39mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to Y39; S2 to L40; G3 to E41; E4 to L42; P5 to A43; G6 to S44; Q7 toA45; T8 to V46; S9 to K47; V10 to E48; A11 to Q49; P12 to Y50; P13 toP51; P14 to G52; E15 to I53; E16 to E54; V17 to I55; E18 to E56; P19 toS57; G20 to R58; S21 to L59; G22 to G60; V23 to G61; R24 to T62; I25 toG63; V26 to A64; V27 to F65; E28 to E66; Y29 to I67; C30 to E68; E31 toI69; P32 to N70; C33 to G71; G34 to Q72; F35 to L73; E36 to V74; A37 toF75; T38 to S76; Y39 to K77; L40 to L78; E41 to E79; L42 to N80; A43 toG81; S44 to G82; A45 to F83; V46 to P84; K47 to Y85; E48 to E86; Q49 toK87; Y50 to D88; P51 to L89; G52 to I90; I53 to E91; E54 to A92; I55 toI93; E56 to R94; S57 to R95; R58 to A96; L59 to S97; G60 to N98; G61 toG99; T62 to E100; G63 to T101; A64 to L102; F65 to E103; E66 to K104;I67 to I105; E68 to T106; I69 to N107; N70 to S108; G71 to R109; Q72 toP110; L73 to P111; V74 to C112; F75 to V113; S76 to I114; K77 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 40mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to L40; S2 to E41; G3 to L42; E4 to A43; P5 to S44; G6 to A45; Q7 toV46; T8 to K47; S9 to E48; V10 to Q49; A11 to Y50; P12 to P51; P13 toG52; P14 to I53; E15 to E54; E16 to I55; V17 to E56; E18 to S57; P19 toR58; G20 to L59; S21 to G60; G22 to G61; V23 to T62; R24 to G63; I25 toA64; V26 to F65; V27 to E66; E28 to I67; Y29 to E68; C30 to I69; E31 toN70; P32 to G71; C33 to Q72; G34 to L73; F35 to V74; E36 to F75; A37 toS76; T38 to K77; Y39 to L78; L40 to E79; E41 to N80; L42 to G81; A43 toG82; S44 to F83; A45 to P84; V46 to Y85; K47 to E86; E48 to K87; Q49 toD88; Y50 to L89; P51 to I90; G52 to E91; I53 to A92; E54 to I93; I55 toR94; E56 to R95; S57 to A96; R58 to S97; L59 to N98; G60 to G99; G61 toE100; T62 to T101; G63 to L102; A64 to E103; F65 to K104; E66 to I105;I67 to T106; E68 to N107; I69 to S108; N70 to R109; G71 to P110; Q72 toP111; L73 to C112; V74 to V113; F75 to I114; S76 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 41mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E41; S2 to L42; G3 to A43; E4 to S44; P5 to A45; G6 to V46; Q7 toK47; T8 to E48; S9 to Q49; V10 to Y50; A11 to P51; P12 to G52; P13 toI53; P14 to E54; E15 to I55; E16 to E56; V17 to S57; E18 to R58; P19 toL59; G20 to G60; S21 to G61; G22 to T62; V23 to G63; R24 to A64; I25 toF65; V26 to E66; V27 to I67; E28 to E68; Y29 to I69; C30 to N70; E31 toG71; P32 to Q72; C33 to L73; G34 to V74; F35 to F75; E36 to S76; A37 toK77; T38 to L78; Y39 to E79; L40 to N80; E41 to G81; L42 to G82; A43 toF83; S44 to P84; A45 to Y85; V46 to E86; K47 to K87; E48 to D88; Q49 toL89; Y50 to I90; P51 to E91; G52 to A92; I53 to I93; E54 to R94; I55 toR95; E56 to A92; S57 to S97; R58 to N98; L59 to G99; G60 to E100; G61 toT101; T62 to L102; G63 to E103; A64 to K104; F65 to I105; E66 to T106;I67 to N107; E68 to S108; I69 to R109; N70 to P110; G71 to P111; Q72 toC112; L73 to V113; V74 to I114; F75 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 42mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to L42; S2 to A43; G3 to S44; E4 to A45; P5 to V46; G6 to K47; Q7 toE48; T8 to Q49; S9 to Y50; V10 to P51; A11 to G52; P12 to I53; P13 toE54; P14 to I55; E15 to E56; E16 to S57; V17 to R58; E18 to L59; P19 toG60; G20 to G61; S21 to T62; G22 to G63; V23 to A64; R24 to F65; I25 toE66; V26 to I67; V27 to E68; E28 to I69; Y29 to N70; C30 to G71; E31 toQ72; P32 to L73; C33 to V74; G34 to F75; F35 to S76; E36 to K77; A37 toL78; T38 to E79; Y39 to N80; L40 to G81; E41 to G82; L42 to F83; A43 toP84; S44 to Y85; A45 to E86; V46 to K87; K47 to D88; E48 to L89; Q49 toI90; Y50 to E91; P51 to A92; G52 to I93; I53 to R94; E54 to R95; I55 toA96; E56 to S97; S57 to N98; R58 to G99; L59 to E100; G60 to T101; G61to L102; T62 to E103; G63 to K104; A64 to I105; F65 to T106; E66 toN107; I67 to S108; E68 to R109; I69 to P110; N70 to P111; G71 to C112;Q72 to V113; L73 to I114; V74 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 43mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to A43; S2 to S44; G3 to A45; E4 to V46; P5 to K47; G6 to E48; Q7 toQ49; T8 to Y50; S9 to P51; V10 to G52; A11 to I53; P12 to E54; P13 toI55; P14 to E56; E15 to S57; E16 to R58; V17 to L59; E18 to G60; P19 toG61; G20 to T62; S21 to G63; G22 to A64; V23 to F65; R24 to E66; I25 toI67; V26 to E68; V27 to I69; E28 to N70; Y29 to G71; C30 to Q72; E31 toL73; P32 to V74; C33 to F75; G34 to S76; F35 to K77; E36 to L78; A37 toE79; T38 to N80; Y39 to G81; L40 to G82; E41 to F83; L42 to P84; A43 toY85; S44 to E86; A45 to K87; V46 to D88; K47 to L89; E48 to I90; Q49 toE91; Y50 to A92; P51 to I93; G52 to R94; I53 to R95; E54 to A96; I55 toS97; E56 to N98; S57 to G99; R58 to E100; L59 to T101; G60 to L102; G61to E103; T62 to K104; G63 to I105; A64 to T106; F65 to N107; E66 toS108; I67 to R109; E68 to P110; I69 to P111; N70 to C112; G71 to V113;Q72 to I114; L73 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 44mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to S44; S2 to A45; G3 to V46; E4 to K47; P5 to E48; G6 to Q49; Q7 toY50; T8 to P51; S9 to G52; V10 to I53; A11 to E54; P12 to I55; P13 toE56; P14 to S57; E15 to R58; E16 to L59; V17 to G60; E18 to G61; P19 toT62; G20 to G63; S21 to A64; G22 to F65; V23 to E66; R24 to I67; I25 toE68; V26 to I69; V27 to N70; E28 to G71; Y29 to Q72; C30 to L73; E31 toV74; P32 to F75; C33 to S76; G34 to K77; F35 to L78; E36 to E79; A37 toN80; T38 to G81; Y39 to G82; L40 to F83; E41 to P84; L42 to Y85; A43 toE86; S44 to K87; A45 to D88; V46 to L89; K47 to I90; E48 to E91; Q49 toA92; Y50 to I93; P51 to R94; G52 to R95; I53 to A96; E54 to S97; I55 toN98; E56 to G99; S57 to E100; R58 to T101; L59 to L102; G60 to E103; G61to K104; T62 to I105; G63 to T106; A64 to N107; F65 to S108; E66 toR109; I67 to P110; E68 to P111; I69 to C112; N70 to V113; G71 to I114;Q72 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 45mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to A45; S2 to V46; G3 to K47; E4 to E48; P5 to Q49; G6 to Y50; Q7 toP51; T8 to G52; S9 to I53; V10 to E54; A11 to I55; P12 to E56; P13 toS57; P14 to R58; E15 to L59; E16 to G60; V17 to G61; E18 to T62; P19 toG63; G20 to A64; S21 to F65; G22 to E66; V23 to I67; R24 to E68; I25 toI69; V26 to N70; V27 to G71; E28 to Q72; Y29 to L73; C30 to V74; E31 toF75; P32 to S76; C33 to K77; G34 to L78; F35 to E79; E36 to N80; A37 toG81; T38 to G82; Y39 to F83; L40 to P84; E41 to Y85; L42 to E86; A43 toK87; S44 to D88; A45 to L89; V46 to I90; K47 to E91; E48 to E92; Q49 toI93; Y50 to R94; P51 to R95; G52 to A96; I53 to S97; E54 to N98; I55 toG99; E56 to E100; S57 to T101; R58 to L102; L59 to E103; G60 to K104;G61 to K105; T62 to T106; G63 to N107; A64 to S108; F65 to R109; E66 toP110; I67 to P111; E68 to C112; I69 to V113; N70 to I114; G71 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 46mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to V46; S2 to K47; G3 to E48; E4 to Q49; P5 to Y50; G6 to P51; Q7 toG52; T8 to I53; S9 to E54; V10 to I55; A11 to E56; P12 to S57; P13 toR58; P14 to L59; E15 to G60; E16 to G61; V17 to T62; E18 to G63; P19 toA64; G20 to F65; S21 to E66; G22 to I67; V23 to E68; R24 to I69; I25 toN70; V26 to G71; V27 to Q72; E28 to L73; Y29 to V74; C30 to F75; E31 toS76; P32 to K77; C33 to L78; G34 to E79; F35 to N80; E36 to G81; A37 toG82; T38 to F83; Y39 to P84; L40 to Y85; E41 to E86; L42 to K87; A43 toD88; S44 to L89; A45 to I90; V46 to E91; K47 to A92; E48 to I93; Q49 toR94; Y50 to R95; P51 to A96; G52 to S97; I53 to N98; E54 to G99; I55 toE100; E56 to T101; S57 to L102; R58 to E103; L59 to K104; G60 to I105;G61 to T106; T62 to N107; G63 to S108; A64 to R109; F65 to P110; E66 toP111; I67 to C112; E68 to V113; I69 to I114; N70 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 47mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to K47; S2 to E48; G3 to Q49; E4 to Y50; P5 to P51; G6 to G52; Q7 toI53; T8 to E54; S9 to I55; V10 to E56; A11 to S57; P12 to R58; P13 toL59; P14 to G60; E15 to G61; E16 to T62; V17 to G63; E18 to A64; P19 toF65; G20 to E66; S21 to I67; G22 to E68; V23 to I69; R24 to N70; I25 toG71; V26 to Q72; V27 to L73; E28 to V74; Y29 to F75; C30 to S76; E31 toK77; P32 to L78; C33 to E79; G34 to N80; F35 to G81; E36 to G82; A37 toF83; T38 to P84; Y39 to Y85; L40 to E86; E41 to K87; L42 to D88; A43 toL89; S44 to I90; A45 to E91; V46 to A92; K47 to I93; E48 to R94; Q49 toR95; Y50 to A96; P51 to S97; G52 to N98; I53 to G99; E54 to E100; I55 toT101; E56 to L102; S57 to E103; R58 to K104; L59 to I105; G60 to T106;G61 to N107; T62 to S108; G63 to R109; A64 to P110; F65 to P111; E66 toC112; I67 to V113; E68 to I114; I69 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 48mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E48; S2 to Q49; G3 to Y50; E4 to P51; P5 to G52; G6 to I53; Q7 toE54; T8 to I55; S9 to E56; V10 to S57; A11 to R58; P12 to L59; P13 toG60; P14 to G61; E15 to T62; E16 to G63; V17 to A64; E18 to F65; P19 toE66; G20 to I67; S21 to E68; G22 to I69; V23 to N70; R24 to G71; I25 toQ72; V26 to L73; V27 to V74; E28 to F75; Y29 to S76; C30 to K77; E31 toL78; P32 to E79; C33 to N80; G34 to G81; F35 to G82; E36 to F83; A37 toP84; T38 to Y85; Y39 to E86; L40 to K87; E41 to D88; L42 to L89; A43 toI90; S44 to E91; A45 to A92; V46 to I93; K47 to R94; E48 to R95; Q49 toA96; Y50 to S97; P51 to N98; G52 to G99; I53 to E100; E54 to T101; I55to L102; E56 to E103; S57 to K104; R58 to I105; L59 to T106; G60 toN107; G61 to S108; T62 to R109; G63 to P110; A64 to P111; F65 to C112;E66 to V113; I67 to I114; E68 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 49mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to Q49; S2 to Y50; G3 to P51; E4 to G52; P5 to I53; G6 to E54; Q7 toI55; T8 to E56; S9 to S57; V10 to R58; A11 to L59; P12 to G60; P13 toG61; P14 to T62; E15 to G63; E16 to A64; V17 to F65; E18 to E66; P19 toI67; G20 to E68; S21 to I69; G22 to N70; V23 to G71; R24 to Q72; I25 toL73; V26 to V74; V27 to F75; E28 to S76; Y29 to K77; C30 to L78; E31 toE79; P32 to N80; C33 to G81; G34 to G82; F35 to F83; E36 to P84; A37 toY85; T38 to E86; Y39 to K87; L40 to D88; E41 to L89; L42 to I90; A43 toE91; S44 to A92; A45 to I93; V46 to R94; K47 to R95; E48 to A96; Q49 toS97; Y50 to N98; P51 to G99; G52 to E100; I53 to T101; E54 to L102; I55to E103; E56 to K104; S57 to I105; R58 to T106; L59 to N107; G60 toS108; G61 to R109; T62 to P110; G63 to P111; A64 to C112; F65 to V113;E66 to I114; I67 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 50mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to Y50; S2 to P51; G3 to G52; E4 to I53; P5 to E54; G6 to I55; Q7 toE56; T8 to S57; S9 to R58; V10 to L59; A11 to G60; P12 to G61; P13 toT62; P14 to G63; E15 to A64; E16 to F65; V17 to E66; E18 to I67; P19 toE68; G20 to I69; S21 to N70; G22 to G71; V23 to Q72; R24 to L73; I25 toV74; V26 to F75; V27 to S76; E28 to K77; Y29 to L78; C30 to E79; E31 toN80; P32 to G81; C33 to G82; G34 to F83; F35 to P84; E36 to Y85; A37 toE86; T38 to K87; Y39 to D88; L40 to L89; E41 to I90; L42 to E91; A43 toA92; S44 to I93; A45 to R94; V46 to R95; K47 to A96; E48 to S97; Q49 toN98; Y50 to G99; P51 to E100; G52 to T101; I53 to L102; E54 to E103; I55to K104; E56 to I105; S57 to T106; R58 to N107; L59 to S108; G60 toR109; G61 to P110; T62 to P111; G63 to C112; A64 to V113; F65 to I114;E66 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 51mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to P51; S2 to G52; G3 to I53; E4 to E54; P5 to I55; G6 to E56; Q7 toS57; T8 to R58; S9 to L59; V10 to G60; A11 to G61; P12 to T62; P13 toG63; P14 to A64; E15 to F65; E16 to E66; V17 to I67; E18 to E68; P19 toI69; G20 to N70; S21 to G71; G22 to Q72; V23 to L73; R24 to V74; I25 toF75; V26 to S76; V27 to K77; E28 to L78; Y29 to E79; C30 to N80; E31 toG81; P32 to G82; C33 to F83; G34 to P84; F35 to Y85; E36 to E86; A37 toK87; T38 to D88; Y39 to L89; L40 to I90; E41 to E91; L42 to A92; A43 toI93; S44 to R94; A45 to R95; V46 to A96; K47 to S97; E48 to N98; Q49 toG99; Y50 to E100; P51 to T101; G52 to L102; I53 to E103; E54 to K104;I55 to I105; E56 to T106; S57 to N107; R58 to S108; L59 to R109; G60 toP110; G61 to P111; T62 to C112; G63 to V113; A64 to I114; F65 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 52mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to G52; S2 to I53; G3 to E54; E4 to I55; P5 to E56; G6 to S57; Q7 toR58; T8 to L59; S9 to G60; V10 to G61; A11 to T62; P12 to G63; P13 toA64; P14 to F65; E15 to E66; E16 to I67; V17 to E68; E18 to I69; P19 toN70; G20 to G71; S21 to Q72; G22 to L73; V23 to V74; R24 to F75; I25 toS76; V26 to K77; V27 to L78; E28 to E79; Y29 to N80; C30 to G81; E31 toG82; P32 to F83; C33 to P84; G34 to Y85; F35 to E86; E36 to K87; A37 toD88; T38 to L89; Y39 to I90; L40 to E91; E41 to A92; L42 to I93; A43 toR94; S44 to R95; A45 to A96; V46 to S97; K47 to N98; E48 to G99; Q49 toE100; Y50 to T101; P51 to L102; G52 to E103; I53 to K104; E54 to I105;I55 to T106; E56 to N107; S57 to S108; R58 to R109; L59 to P110; G60 toP111; G61 to C112; T62 to V113; G63 to I114; A64 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 53mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to I53; S2 to E54; G3 to I55; E4 to E56; P5 to S57; G6 to R58; Q7 toL59; T8 to G60; S9 to G61; V10 to T62; A11 to G63; P12 to A64; P13 toF65; P14 to E66; E15 to I67; E16 to E68; V17 to I69; E18 to N70; P19 toG71; G20 to Q72; S21 to L73; G22 to V74; V23 to F75; R24 to S76; I25 toK77; V26 to L78; V27 to E79; E28 to N80; Y29 to G81; C30 to G82; E31 toF83; P32 to P84; C33 to Y85; G34 to E86; F35 to K87; E36 to D88; A37 toL89; T38 to I90; Y39 to E91; L40 to A92; E41 to I93; L42 to R94; A43 toR95; S44 to A96; A45 to S97; V46 to N98; K47 to G99; E48 to E100; Q49 toT101; Y50 to L102; P51 to E103; G52 to K104; I53 to I105; E54 to T106;I55 to N107; E56 to S108; S57 to R109; R58 to P110; L59 to P111; G60 toC112; G61 to V113; T62 to I114; G63 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 54mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E54; S2 to I55; G3 to E56; E4 to S57; P5 to R58; G6 to L59; Q7 toG60; T8 to G61; S9 to T62; V10 to G63; A11 to A64; P12 to F65; P13 toE66; P14 to I67; E15 to E68; E16 to I69; V17 to N70; E18 to G71; P19 toQ72; G20 to L73; S21 to V74; G22 to F75; V23 to S76; R24 to K77; I25 toL78; V26 to E79; V27 to N80; E28 to G81; Y29 to G82; C30 to F83; E31 toP84; P32 to Y85; C33 to E86; G34 to K87; F35 to D88; E36 to L89; A37 toI90; T38 to E91; Y39 to A92; L40 to I93; E41 to R94; L42 to R95; A43 toA96; S44 to S97; A45 to N98; V46 to G99; K47 to E100; E48 to T101; Q49to L102; Y50 to E103; P51 to K104; G52 to I105; I53 to T106; E54 toN107; I55 to S108; E56 to R109; S57 to P110; R58 to P111; L59 to C112;G60 to V113; G61 to I114; T62 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 55mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to I55; S2 to E56; G3 to S57; E4 to R58; P5 to L59; G6 to G60; Q7 toG61; T8 to T62; S9 to G63; V10 to A64; A11 to F65; P12 to E66; P13 toI67; P14 to E68; E15 to I69; E16 to N70; V17 to G71; E18 to Q72; P19 toL73; G20 to V74; S21 to F75; G22 to S76; V23 to K77; R24 to L78; I25 toE79; V26 to N80; V27 to G81; E28 to G82; Y29 to F83; C30 to P84; E31 toY85; P32 to E86; C33 to K87; G34 to D88; F35 to L89; E36 to I90; A37 toE91; T38 to A92; Y39 to I93; L40 to R94; E41 to R95; L42 to A96; A43 toS97; S44 to N98; A45 to G99; V46 to E100; K47 to T101; E48 to L102; Q49to E103; Y50 to K104; P51 to I105; G52 to T106; I53 to N107; E54 toS108; I55 to R109; E56 to P110; S57 to P111; R58 to C112; L59 to V113;G60 to I114; G61 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 56mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E56; S2 to S57; G3 to R58; E4 to L59; P5 to G60; G6 to G61; Q7 toT62; T8 to G63; S9 to A64; V10 to F65; A11 to E66; P12 to I67; P13 toE68; P14 to I69; E15 to N70; E16 to G71; V17 to Q72; E18 to L73; P19 toV74; G20 to F75; S21 to S76; G22 to K77; V23 to L78; R24 to E79; I25 toN80; V26 to G81; V27 to G82; E28 to F83; Y29 to P84; C30 to Y85; E31 toE86; P32 to K87; C33 to D88; G34 to L89; F35 to I90; E36 to E91; A37 toA92; T38 to I93; Y39 to R94; L40 to R95; E41 to A96; L42 to S97; A43 toN98; S44 to G99; A45 to E100; V46 to T100; K47 to L102; E48 to E103; Q49to K104; Y50 to I105; P51 to T106; G52 to N107; I53 to S108; E54 toR109; I55 to P110; E56 to P111; S57 to C112; R58 to V113; L59 to I114;G60 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 57mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to S57; S2 to R58; G3 to L59; E4 to G60; P5 to G61; G6 to T62; Q7 toG63; T8 to A64; S9 to F65; V10 to E66; A11 to I67; P12 to E68; P13 toI69; P14 to N70; E15 to G71; E16 to Q72; V17 to L73; E18 to V74; P19 toF75; G20 to S76; S21 to K77; G22 to L78; V23 to E79; R24 to N80; I25 toG81; V26 to G82; V27 to F83; E28 to P84; Y29 to Y85; C30 to E86; E31 toK87; P32 to D88; C33 to L89; G34 to I90; F35 to E91; E36 to A92; A37 toI93; T38 to R94; Y39 to R95; L40 to A96; E41 to S97; L42 to N98; A43 toG99; S44 to E100; A45 to T100; V46 to L102; K47 to E103; E48 to K104;Q49 to I105; Y50 to T106; P51 to N107; G52 to S108; I53 to R109; E54 toP110; I55 to P111; E56 to C112; S57 to V113; R58 to I114; L59 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 58mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to R58; S2 to L59; G3 to G60; E4 to G61; P5 to T62; G6 to G63; Q7 toA64; T8 to F65; S9 to E66; V10 to I67; A11 to E68; P12 to I69; P13 toN70; P14 to G71; E15 to Q72; E16 to L73; V17 to V74; E18 to F75; P19 toS76; G20 to K77; S21 to L78; G22 to E79; V23 to N80; R24 to G81; I25 toG82; V26 to F83; V27 to P84; E28 to Y85; Y29 to E86; C30 to K87; E31 toE88; P32 to L89; C33 to I90; G34 to E91; F35 to A92; E36 to I93; A37 toR94; T38 to R95; Y39 to A96; L40 to S97; E1 to N98; L42 to G99; A43 toE100; S44 to T101; A45 to L102; V46 to E103; K47 to K104; E48 to I105;Q49 to T106; Y50 to N107; P51 to S108; G52 to R109; I53 to P110; E54 toP111; I55 to C112; E56 to V113; S57 to I114; R58 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 59mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to L59; S2 to G60; G3 to G61; E4 to T62; P5 to G63; G6 to A64; Q7 toF65; T8 to E66; S9 to I67; V10 to E68; A11 to I69; P12 to N70; P13 toG71; P14 to Q72; E15 to L73; E16 to V74; V17 to F75; E18 to S76; P19 toK77; G20 to L78; S21 to E79; G22 to N80; V23 to G81; R24 to G82; I25 toF83; V26 to P84; V27 to Y85; E28 to E86; Y29 to K87; C30 to D88; E31 toL89; P32 to I90; C33 to E91; G34 to A92; F35 to I93; E36 to R94; A37 toR95; T38 to A96; Y39 to S97; L40 to N98; E41 to G99; L42 to E100; A43 toT101; S44 to L102; A45 to E103; V46 to K104; K47 to I105; E48 to T106;Q49 to N107; Y50 to S108; P51 to R109; G52 to P110; I53 to P111; E54 toC112; I55 to V113; E56 to I114; S57 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 60mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to G60; S2 to G61; G3 to T62; E4 to G63; P5 to A64; G6 to F65; Q7 toE66; T8 to I67; S9 to E68; V10 to I69; A11 to N70; P12 to G71; P13 toQ72; P14 to L73; E15 to V74; E16 to F75; V17 to S76; E18 to K77; P19 toL78; G20 to E79; S21 to N80; G22 to G81; V23 to G82; R24 to F83; I25 toP84; V26 to Y85; V27 to E86; E28 to K87; Y29 to D88; C30 to L89; E31 toI90; P32 to E91; C33 to A92; G34 to I93; F35 to R94; E36 to R95; A37 toA96; T38 to S97; Y39 to N98; L40 to G99; E41 to E100; L42 to T101; A43to L102; S44 to E103; A45 to K104; V46 to I105; K47 to T106; E48 toN107; Q49 to S108; Y50 to R109; P51 to P110; G52 to P111; I53 to C112;E54 to V113; I55 to I114; E56 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 61mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to G61; S2 to T62; G3 to G63; E4 to A64; P5 to F65; G6 to E66; Q7 toI67; T8 to E68; S9 to I69; V10 to N70; A11 to G71; P12 to Q72; P13 toL73; P14 to V74; E15 to F75; E16 to S76; V17 to K77; E18 to L78; P19 toE79; G20 to N80; S21 to G81; G22 to G82; V23 to F83; R24 to P84; I25 toY85; V26 to E86; V27 to K87; E28 to D88; Y29 to L89; C30 to I90; E31 toE91; P32 to A92; C33 to I93; G34 to R94; F35 to R95; E36 to A96; A37 toS97; T38 to N98; Y39 to G99; L40 to E100; E41 to T101; L42 to L102; A43to E103; S44 to K104; A45 to I105; V46 to T106; K47 to N107; E48 toS108; Q49 to R109; Y50 to P110; P51 to P111; G52 to C112; I53 to V113;E54 to I114; I55 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 62mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to T62; S2 to G63; G3 to A64; E4 to F65; P5 to E66; G6 to I67; Q7 toE68; T8 to I69; S9 to N70; V10 to G71; A11 to Q72; P12 to L73; P13 toV74; P14 to F75; E15 to S76; E16 to K77; V17 to L78; E18 to E79; P19 toN80; G20 to G81; S21 to G82; G22 to F83; V23 to P84; R24 to Y85; I25 toE86; V26 to K87; V27 to D88; E28 to L89; Y29 to I90; C30 to E91; E31 toA92; P32 to I93; C33 to R94; G34 to R95; F35 to A96; E36 to S97; A37 toN98; T38 to G99; Y39 to E100; L40 to T101; E41 to L102; L42 to E103; A43to K104; S44 to I105; A45 to T106; V46 to N107; K47 to S108; E48 toR109; Q49 to P110; Y50 to P111; P51 to C112; G52 to V113; I53 to I114;E54 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 63mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to G63; S2 to A64; G3 to F65; E4 to E66; P5 to I67; G6 to E68; Q7 toI69; T8 to N70; S9 to G71; V10 to Q72; A11 to L73; P12 to V74; P13 toF75; P14 to S76; E15 to K77; E16 to L78; V17 to E79; E18 to N80; P19 toG81; G20 to G82; S21 to F83; G22 to P84; V23 to Y85; R24 to E86; I25 toK87; V26 to D88; V27 to L89; E28 to I90; Y29 to E91; C30 to A92; E31 toI93; P32 to R94; C33 to R95; G34 to A96; F35 to S97; E36 to N98; A37 toG99; T38 to E100; Y39 to T101; L40 to L102; E41 to E103; L42 to K104;A43 to I105; S44 to T106; A45 to N107; V46 to S108; K47 to R109; E48 toP110; Q49 to P111; Y50 to C112; P51 to V113; G52 to I114; I53 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 64mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to A64; S2 to F65; G3 to E66; E4 to I67; P5 to E68; G6 to I69; Q7 toN70; T8 to G71; S9 to Q72; V10 to L73; A11 to V74; P12 to F75; P13 toS76; P14 to K77; E15 to L78; E16 to E79; V17 to N80; E18 to G81; P19 toG82; G20 to F83; S21 to P84; G22 to Y85; V23 to E86; R24 to K87; I25 toD88; V26 to L89; V27 to I90; E28 to E91; Y29 to A92; C30 to I93; E31 toR94; P32 to R95; C33 to A96; G34 to S97; F35 to N98; E36 to G99; A37 toE100; T38 to T101; Y39 to L102; L40 to E103; E41 to K104; L42 to I105;A43 to T106; S44 to N107; A45 to S108; V46 to R109; K47 to P110; E48 toP111; Q49 to C112; Y50 to V113; P51 to I114; G52 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 65mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to F65; S2 to E66; G3 to I67; E4 to E68; P5 to I69; G6 to N70; Q7 toG71; T8 to Q72; S9 to L73; V10 to V74; A11 to F75; P12 to S76; P13 toK77; P14 to L78; E15 to E79; E16 to N80; V17 to G81; E18 to G82; P19 toF83; G20 to P84; S21 to Y85; G22 to E86; V23 to K87; R24 to D88; I25 toL89; V26 to I90; V27 to E91; E28 to A92; Y29 to I93; C30 to R94; E31 toR95; P32 to A96; C33 to S97; G34 to N98; F35 to G99; E36 to E100; A37 toT101; T38 to L102; Y39 to E103; L40 to K104; E41 to I105; L42 to T106;A43 to N107; S44 to S108; A45 to R109; V46 to P110; K47 to P111; E48 toC112; Q49 to V113; Y50 to I114; P51 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 66mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E66; S2 to I67; G3 to E68; E4 to I69; P5 to N70; G6 to G71; Q7 toQ72; T8 to L73; S9 to V74; V10 to F75; A11 to S76; P12 to K77; P13 toL78; P14 to E79; E15 to N80; E16 to G81; V17 to G82; E18 to F83; P19 toP84; G20 to Y85; S21 to E86; G22 to K87; V23 to D88; R24 to L89; I25 toI90; V26 to E91; V27 to A92; E28 to I93; Y29 to R94; C30 to R95; E31 toA96; P32 to S97; C33 to N98; G34 to G99; F35 to E100; E36 to T101; A37to L102; T38 to E103; Y39 to K104; L40 to I105; E41 to T106; L42 toN107; A43 to S108; S44 to R109; A45 to P110; V46 to P111; K47 to C112;E48 to V113; Q49 to I114; Y50 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 67mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to I67; S2 to E68; G3 to I69; E4 to N70; P5 to G71; G6 to Q72; Q7 toL73; T8 to V74; S9 to F75; V10 to S76; A11 to K77; P12 to L78; P13 toE79; P14 to N80; E15 to G81; E16 to G82; V17 to F83; E18 to P84; P19 toY85; G20 to E86; S21 to K87; G22 to D88; V23 to L89; R24 to I90; I25 toE91; V26 to A92; V27 to I93; E28 to R94; Y29 to R95; C30 to A96; E31 toS97; P32 to N98; C33 to G99; G34 to E100; F35 to T101; E36 to L102; A37to E103; T38 to K104; Y39 to I105; L40 to T106; E41 to N107; L42 toS108; A43 to R109; S44 to P110; A45 to P111; V46 to C112; K47 to V113;E48 to I114; Q49 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 68mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E68; S2 to I69; G3 to N70; E4 to G71; P5 to Q72; G6 to L73; Q7 toV74; T8 to F75; S9 to S76; V10 to K77; A11 to L78; P12 to E79; P13 toN80; P14 to G81; E15 to G82; E16 to F83; V17 to P84; E18 to Y85; P19 toE86; G20 to K87; S21 to D88; G22 to L89; V23 to I90; R24 to E91; I25 toA92; V26 to I93; V27 to R94; E28 to R95; Y29 to A96; C30 to S97; E31 toN98; P32 to G99; C33 to E100; G34 to T101; F35 to L102; E36 to E103; A37to K104; T38 to I105; Y39 to T106; L40 to N107; E41 to S108; L42 toR109; A43 to P110; S44 to P111; A45 to C112; V46 to V113; K47 to I114;E48 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 69mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to I69; S2 to N70; G3 to G71; E4 to Q72; P5 to L73; G6 to V74; Q7 toF75; T8 to S76; S9 to K77; V10 to L78; A11 to E79; P12 to N80; P13 toG81; P14 to G82; E15 to F83; E16 to P84; V17 to Y85; E18 to E86; P19 toK87; G20 to D88; S21 to L89; G22 to I90; V23 to E91; R24 to A92; I25 toI93; V26 to R94; V27 to R95; E28 to A96; Y29 to S97; C30 to N98; E31 toG99; P32 to E100; C33 to T101; G34 to L102; F35 to E103; E36 to K104;A37 to I105; T38 to T106; Y39 to N107; L40 to S108; E41 to R109; L42 toP110; A43 to P111; S44 to C112; A45 to V113; V46 to I114; K47 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 70mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to N70; S2 to G71; G3 to Q72; E4 to L73; P5 to V74; G6 to F75; Q7 toS76; T8 to K77; S9 to L78; V10 to E79; A11 to N80; P12 to G81; P13 toG82; P14 to F83; E15 to P84; E16 to Y85; V17 to E86; E18 to K87; P19 toD88; G20 to L89; S21 to I90; G22 to E91; V23 to A92; R24 to I93; I25 toR94; V26 to R95; V27 to A96; E28 to S97; Y29 to N98; C30 to G99; E31 toE100; P32 to T101; C33 to L102; G34 to E103; F35 to K104; E36 to I105;A37 to T106; T38 to N107; Y39 to S108; L40 to R109; E41 to P110; L42 toP111; A43 to C112; S44 to V113; A45 to I114; V46 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 71mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to G71; S2 to Q72; G3 to L73; E4 to V74; P5 to F75; G6 to S76; Q7 toK77; T8 to L78; S9 to E79; V10 to N80; A11 to G81; P12 to G82; P13 toF83; P14 to P84; E15 to Y85; E16 to E86; V17 to K87; E18 to D88; P19 toL89; G20 to I90; S21 to E91; G22 to A92; V23 to I93; R24 to R94; I25 toR95; V26 to A96; V27 to S97; E28 to N98; Y29 to G99; C30 to E100; E31 toT101; P32 to L102; C33 to E103; G34 to K104; F35 to I105; E36 to T106;A37 to N107; T38 to S108; Y39 to R109; L40 to P110; E41 to P111; L42 toC112; A43 to V113; S44 to I114; A45 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 72mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to Q72; S2 to L73; G3 to V74; E4 to F75; P5 to S76; G6 to K77; Q7 toL78; T8 to E79; S9 to N80; V10 to G81; A11 to G82; P12 to F83; P13 toP84; P14 to Y85; E15 to E86; E16 to K87; V17 to D88; E18 to L89; P19 toI90; G20 to E91; S21 to A92; G22 to I93; V23 to R94; R24 to R95; I25 toA96; V26 to S97; V27 to N98; E28 to G99; Y29 to E100; C30 to T101; E31to L102; P32 to E103; C33 to K104; G34 to I105; F35 to T106; E36 toN107; A37 to S108; T38 to R109; Y39 to P110; L40 to P111; E41 to C112;L42 to V113; A43 to I114; S44 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 73mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to L73; S2 to V74; G3 to F75; E4 to S76; P5 to K77; G6 to L78; Q7 toE79; T8 to N80; S9 to G81; V10 to G82; A11 to F83; P12 to P84; P13 toY85; P14 to E86; E15 to K87; E16 to D88; V17 to L89; E18 to I90; P19 toE91; G20 to A92; S21 to I93; G22 to R94; V23 to R95; R24 to A96; I25 toS97; V26 to N98; V27 to G99; E28 to E100; Y29 to T101; C30 to L102; E31to E103; P32 to K104; C33 to I105; G34 to T106; F35 to N107; E36 toS108; A37 to R109; T38 to P110; Y39 to P111; L40 to C112; E41 to V113;L42 to I114; A43 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 74mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to V74; S2 to F75; G3 to S76; E4 to K77; P5 to L78; G6 to E79; Q7 toN80; T8 to G81; S9 to G82; V10 to F83; A11 to P84; P12 to Y85; P13 toE86; P14 to K87; E15 to D88; E16 to L89; V17 to I90; E18 to E91; P19 toA92; G20 to I93; S21 to R94; G22 to R95; V23 to A96; R24 to S97; I25 toN98; V26 to G99; V27 to E100; E28 to T100; Y29 to L102; C30 to E103; E31to K104; P32 to I105; C33 to T106; G34 to N107; F35 to S108; E36 toR109; A37 to P110; T38 to P111; Y39 to C112; L40 to V113; E41 to I114;L42 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 75mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to F75; S2 to S76; G3 to K77; E4 to L78; P5 to E79; G6 to N80; Q7 toG81; T8 to G82; S9 to F83; V10 to P84; A11 to Y85; P12 to E86; P13 toK87; P14 to D88; E15 to L89; E16 to I90; V17 to E91; E18 to A92; P19 toI93; G20 to R94; S21 to R95; G22 to A96; V23 to S97; R24 to N98; I25 toG99; V26 to E100; V27 to T101; E28 to L102; Y29 to E103; C30 to K104;E31 to I105; P32 to T106; C33 to N107; G34 to S108; F35 to R109; E36 toP110; A37 to P111; T38 to C112; Y39 to V113; L40 to I114; E41 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 76mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to S76; S2 to K77; G3 to L78; E4 to E79; P5 to N80; G6 to G81; Q7 toG82; T8 to F83; S9 to P84; V10 to Y85; A11 to E86; P12 to K87; P13 toD88; P14 to L89; E15 to I90; E16 to E91; V17 to A92; E18 to I93; P19 toR94; G20 to R95; S21 to A96; G22 to S97; V23 to N98; R24 to G99; I25 toE100; V26 to T101; V27 to L102; E28 to E103; Y29 to K104; C30 to I105;E31 to T106; P32 to N107; C33 to S108; G34 to R109; F35 to P110; E36 toP111; A37 to C112; T38 to V113; Y39 to I114; L40 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 77mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to K77; S2 to L78; G3 to E79; E4 to N80; P5 to G81; G6 to G82; Q7 toF83; T8 to P84; S9 to Y85; V10 to E86; A11 to K87; P12 to D88; P13 toL89; P14 to I90; E15 to E91; E16 to A92; V17 to I93; E18 to R94; P19 toR95; G20 to A96; S21 to S97; G22 to N98; V23 to G99; R24 to E100; I25 toT101; V26 to L102; V27 to E103; E28 to K104; Y29; I105; C30 to T106; E31to N107; P32 to S108; C33 to R109; G34 to P110; F35 to P111; E36 toC112; A37 to V113; T38 to I114; Y39 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 78mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to L78; S2 to E79; G3 to N80; E4 to G81; P5 to G82; G6 to F83; Q7 toP84; T8 to Y85; S9 to E86; V10 to K87; A11 to D88; P12 to L89; P13 toI90; P14 to E91; E15 to A92; E16 to I93; V17 to R94; E18 to R95; P19 toA96; G20 to S97; S21 to N98; G22 to G99; V23 to E100; R24 to T101; I25to L102; V26 to E103; V27 to K104; E28 to I105; Y29 to T106; C30 toN107; E31 to S108; P32 to R109; C33 to P110; G34 to P111; F35 to C112;E36 to V113; A37 to I114; T38 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 79mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E79; S2 to N80; G3 to G81; E4 to G82; P5 to F83; G6 to P84; Q7 toY85; T8 to E86; S9 to K87; V10 to D88; A11 to L89; P12 to I90; P13 toE91; P14 to A92; E15 to I93; E16 to R94; V17 to R95; E18 to A96; P19 toS97; G20 to N98; S21 to G99; G22 to E100; V23 to T101; R24 to L102; I25to E103; V26 to K104; V27 to I105; E28 to T106; Y29 to N107; C30 toS108; E31 to R109; P32 to P110; C33 to P111; G34 to C112; F35 to V113;E36 to I114; A37 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 80mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to N80; S2 to G81; G3 to G82; E4 to F83; P5 to P84; G6 to Y85; Q7 toE86; T8 to K87; S9 to D88; V10 to L89; A11 to I90; P12 to E91; P13 toA92; P14 to I93; E15 to R94; E16 to R95; V17 to A96; E18 to S97; P19 toN98; G20 to G99; S21 to E100; G22 to T101; V23 to L102; R24 to E103; I25to K104; V26 to I105; V27 to T106; E28 to N107; Y29 to S108; C30 toR109; E31 to P110; P32 to P111; C33 to C112; G34 to V113; F35 to I114;E36 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 81mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to G81; S2 to G82; G3 to F83; E4 to P84; P5 to Y85; G6 to E86; Q7 toK87; T8 to D88; S9 to L89; V10 to I90; A11 to E91; P12 to A92; P13 toI93; P14 to R94; E15 to R95; E16 to A96; V17 to S97; E18 to N98; P19 toG99; G20 to E100; S21 to T101; G22 to L102; V23 to E103; R24 to K104;I25 to I105; V26 to T106; V27 to N107; E28 to S108; Y29 to R109; C30 toP110; E31 to P111; P32 to C112; C33 to V113; G34 to I114; F35 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 82mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to G82; S2 to F83; G3 to P84; E4 to Y85; P5 to E86; G6 to K87; Q7 toD88; T8 to L89; S9 to I90; V10 to E91; A11 to A92; P12 to I93; P13 toR94; P14 to R95; E15 to A96; E16 to S97; V17 to N98; E18 to G99; P19 toE100; G20 to T101; S21 to L102; G22 to E103; V23 to K104; R24 to I105;I25 to T106; V26 to N107; V27 to S108; E28 to R109; Y29 to P110; C30 toP111; E31 to C112; P32 to V113; C33 to I114; G34 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 83mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to F83; S2 to P84; G3 to Y85; E4 to E86; P5 to K87; G6 to D88; Q7 toL89; T8 to I90; S9 to E91; V10 to A92; A11 to I93; P12 to R94; P13 toR95; P14 to A96; E15 to S97; E16 to N98; V17 to G99; E18 to E100; P19 toT101; G20 to L102; S21 to E103; G22 to K104; V23 to I105; R24 to T106;I25 to N107; V26 to S108; V27 to R109; E28 to P110; Y29 to P111; C30 toC112; E31 to V113; P32 to I114; C33 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 84mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to P84; S2 to Y85; G3 to E86; E4 to K87; P5 to D88; G6 to L89; Q7 toI90; T8 to E91; S9 to A92; V10 to I93; A11 to R94; P12 to R95; P13 toA96; P14 to S97; E15 to N98; E16 to G99; V17 to E100; E18 to T101; P19to L102; G20 to E103; S21 to K104; G22 to I105; V23 to T106; R24 toN107; I25 to S108; V26 to R109; V27 to P110; E28 to P111; Y29 to C112;C30 to V113; E31 to I114; P32 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 85mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to Y85; S2 to E86; G3 to K87; E4 to D88; P5 to L89; G6 to I90; Q7 toE91; T8 to A92; S9 to I93; V10 to R94; A11 to R95; P12 to A96; P13 toS97; P14 to N98; E15 to G99; E16 to E100; V17 to T101; E18 to L102; P19to E103; G20 to K104; S21 to I105; G22 to T106; V23 to N107; R24 toS108; I25 to R109; V26 to P110; V27 to P111; E28 to C112; Y29 to V113;C30 to I114; E31 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 86mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E86; S2 to K87; G3 to D88; E4 to L89; P5 to I90; G6 to E91; Q7 toA92; T8 to I93; S9 to R94; V10 to R95; A11 to A96; P12 to S97; P13 toN98; P14 to G99; E15 to E100; E16 to T101; V17 to L102; E18 to E103; P19to K104; G20 to I105; S21 to T106; G22 to N107; V23 to S108; R24 toR109; I25 to P110; V26 to P111; V27 to C112; E28 to V113; Y29 to I114;C30 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 87mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to K87; S2 to D88; G3 to L89; E4 to I90; P5 to E91; G6 to A92; Q7 toI93; T8 to R94; S9 to R95; V10 to A96; A11 to S97; P12 to N98; P13 toG99; P14 to E100; E15 to T101; E16 to L102; V17 to E103; E18 to K104;P19 to I105; G20 to T106; S21 to N107; G22 to S108; V23 to R109; R24 toP110; I25 to P111; V26 to C112; V27 to V113; E28 to I114; Y29 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 88mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to D88; S2 to L89; G3 to I90; E4 to E91; P5 to A92; G6 to I93; Q7 toR94; T8 to R95; S9 to A96; V10 to S97; A11 to N98; P12 to G99; P13 toE100; P14 to T101; E15 to L102; E16 to E103; V17 to K104; E18 to I105;P19 to T106; G20 to N107; S21 to S108; G22 to R109; V23 to P110; R24 toP111; I25 to C112; V26 to V113; V27 to I114; E28 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 89mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to L89; S2 to I90; G3 to E91; E4 to A92; P5 to I93; G6 to R94; Q7 toR95; T8 to A96; S9 to S97; V10 to N98; A11 to G99; P12 to E100; P13 toT101; P14 to L102; E15 to E103; E16 to K104; V17 to I105; E18 to T106;P19 to N107; G20 to S108; S21 to R109; G22 to P110; V23 to P111; R24 toC112; I25 to V113; V26 to I114; V27 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 90mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to I90; S2 to E91; G3 to A92; E4 to I93; P5 to R94; G6 to R95; Q7 toA96; T8 to S97; S9 to N98; V10 to G99; A11 to E100; P12 to T101; P13 toL102; P14 to E103; E15 to K104; E16 to I105; V17 to T106; E18 to N107;P19 to S108; G20 to R109; S21 to P110; G22 to P111; V23 to C112; R24 toV113; I25 to I114; V26 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 91mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E91; S2 to A92; G3 to I93; E4 to R94; P5 to R95; G6 to A96; Q7 toS97; T8 to N98; S9 to G99; V10 to E100; A11 to T101; P12 to L102; P13 toE103; P14 to K104; E15 to I105; E16 to T106; V17 to N107; E18 to S108;P19 to R109; G20 to P110; S21 to P111; G22 to C112; V23 to V113; R24 toI114; I25 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 92mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to A92; S2 to I93; G3 to R94; E4 to R95; P5 to A96; G6 to S97; Q7 toN98; T8 to G99; S9 to E100; V10 to T101; A11 to L102; P12 to E103; P13to K104; P14 to I105; E15 to T106; E16 to N107; V17 to S108; E18 toR109; P19 to P110; G20 to P111; S21 to C112; G22 to V113; V23 to I114;R24 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 93mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to I93; S2 to R94; G3 to R95; E4 to A96; P5 to S97; G6 to N98; Q7 toG99; T8 to E100; S9 to T10; V10 to L102; A11 to E103; P12 to K104; P13to I105; P14 to T106; E15 to N107; E16 to S108; V17 to R109; E18 toP110; P19 to P111; G20 to C112; S21 to V113; G22 to I114; V23 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 94mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to R94; S2 to R95; G3 to A96; E4 to S97; P5 to N98; G6 to G99; Q7 toE100; T8 to T101; S9 to L102; V10 to E103; A11 to K104; P12 to I105; P13to T106; P14 to N107; E15 to S108; E16 to R109; V17 to P110; E18 toP111; P19 to C112; G20 to V113; S21 to I114; G22 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 95mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B): M1 to R95; S2 to A96; G3 to S97;E4 to N98; P5 to G99; G6 to E100; Q7 to T101; T8 to L102; S9 to E103;V10 to K104; A11 to I105; P12 to T106; P13 to N107; P14 to S108; E15 toR109; E16 to P110; V17 to P111; E18 to C112; P19 to V113; G20 to I114;S21 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 96mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to A96; S2 to S97; G3 to N98; E4 to G99; P5 to E100; G6 to T101; Q7to L102; T8 to E103; S9 to K104; V10 to I105; A11 to T106; P12 to N107;P13 to S108; P14 to R109; E15 to P110; E16 to P111; V17 to C112; E18 toV113; P19 to I114; G20 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 97mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to S97; S2 to N98; G3 to G99; E4 to E100; P5 to T101; G6 to L102; Q7to E103; T8 to K104; S9 to I105; V10 to T106; A11 to N107; P12 to S108;P13 to R109; P14 to P110; E15 to P111; E16 to C112; V17 to V113; E18 toI114; P19 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 98mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to N98; S2 to G99; G3 to E100; E4 to T100; P5 to L102; G6 to E103; Q7to K104; T8 to I105; S9 to T106; V10 to N107; A11 to S108; P12 to R109;P13 to P110; P14 to P111; E15 to C112; E16 to V113; V17 to I114; E18 toL115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 99mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to G99; S2 to E100; G3 to T101; E4 to L102; P5 to E103; G6 to K104;Q7 to I105; T8 to T106; S9 to N107; V10 to S108; A11 to R109; P12 toP110; P13 to P111; P14 to C112; E15 to V113; E16 to I114; V17 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 100mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E100; S2 to T101; G3 to L102; E4 to E103; P5 to K104; G6 to I105;Q7 to T106; T8 to N107; S9 to S108; V10 to R109; A11 to P110; P12 toP111; P13 to C112; P14 to V113; E15 to I114; E16 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 101mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to T101; S2 to L102; G3 to E103; E4 to K104; P5 to I105; G6 to T106;Q7 to N107; T8 to S108; S9 to R109; V10 to P110; A11 to P111; P12 toC112; P13 to V113; P14 to I114; E15 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 102mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to L102; S2 to E103; G3 to K104; E4 to I105; P5 to T106; G6 to N107;Q7 to S108; T8 to R109; S9 to P110; V10 to P111; A11 to C112; P12 toV113; P13 to I114; P14 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 103mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to E103; S2 to K104; G3 to I105; E4 to T106; P5 to N107; G6 to S108;Q7 to R109; T8 to P110; S9 to P111; V10 to C112; A11 to V113; P12 toI114; P13 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 104mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to K104; S2 to I105; G3 to T106; E4 to N107; P5 to S108; G6 to R109;Q7 to P110; T8 to P111; S9 to C112; V10 to V113; A11 to I114; P12 toL115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 105mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to I105; S2 to T106; G3 to N107; E4 to S108; P5 to R109; G6 to P110;Q7 to P111; T8 to C112; S9 to V113; V10 to I114; A11 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 106mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to T106; S2 to N107; G3 to S108; E4 to R109; P5 to P110; G6 to P111;Q7 to C112; T8 to V113; S9 to I114; V10 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 107mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to N107; S2 to S108; G3 to R109; E4 to P110; P5 to P111; G6 to C112;Q7 to V113; T8 to I114; S9 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 108mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to S108; S2 to R109; G3 to P110; E4 to P111; P5 to C112; G6 to V113;Q7 to I114; T8 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 109mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to R109; S2 to P110; G3 to P111; E4 to C112; P5 to V113; G6 to I114;Q7 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 110mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to P110; S2 to P111; G3 to C112; E4 to V113; P5 to I114; G6 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 111mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to P111; S2 to C112; G3 to V113; E4 to I114; P5 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 112mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to C112; S2 to V113; G3 to I114; E4 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 113mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to V113; S2 to I114; G3 to L115.

In another preferred embodiment, the isolated polypeptides of thepresent invention comprising or, alternatively, consisting of, one ormore C35 peptide epitopes include the following 114mers (residuescorrespond to SEQ ID NO:2 and FIG. 1B):

M1 to I114; S2 to L115.

Stimulation of CTL and HTL Responses

Much more about the mechanism by which T cells recognize antigens hasbeen elucidated during the past ten years. In accordance with thisunderstanding of the immune system, the present inventors have developedefficacious peptide epitope compositions that induce a therapeutic orprophylactic immune response to certain tumor associated antigens, whenadministered via various art-accepted modalities. Moreover, by use ofthe peptide epitopes of the invention, or by use of combinations ofpeptide epitopes in accordance with the principles disclosed herein,responses can be achieved in significant percentages of anon-genetically biased worldwide population. For an understanding of thevalue and efficacy of the claimed compositions, a brief review ofimmunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs-required for specific binding to HLA antigen molecules have beenidentified.

Furthermore, x-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft of HLA moleculeswhich accommodate, often on an allele-specific basis, residues borne bypeptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol.Biol. 219:277, 1991.) Accordingly, the definition of class I and classII allele-specific HLA binding motifs, or class I or class IIsupermotifs allows identification of regions within a protein that havethe predicted ability to bind particular HLA antigen(s).

Moreover, the correlation of binding affinity with immunogenicity, whichis disclosed herein, is an important factor to be considered whenevaluating candidate peptides. Thus, by a combination of motif searchesof antigenic sequences, and by HLA-peptide binding assays, epitope-basedvaccines have been identified. As appreciated by one in the art, afterdetermining their binding affinity, additional work can be performed toselect, amongst these vaccine peptides, e.g., epitopes can be selectedhaving optional characteristics in terms of population coverage,antigenicity, and immunogenicity, etc.

Various strategies can be utilized to evaluate immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a 51Cr-release assay involvingpeptide sensitized target cells, and/or target cells that generateantigen endogenously.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol.8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997); in thismethod, peptides in incomplete Freund's adjuvant are administeredsubcutaneously to HLA transgenic mice. Several weeks followingimmunization, splenocytes are removed and cultured in vitro in thepresence of test peptide for approximately one week. Peptide-specific Tcells are detected using, e.g., a 51Cr-release assay involving peptidesensitized target cells and target cells expressing endogenouslygenerated antigen.

3) Demonstration of recall T cell responses from individuals exposed tothe disease, such as immune individuals who were effectively treated andrecovered from disease, and/or from actively ill patients (see, e.g.,Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al.,Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997;Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. etal., J. Virol. 71:6011, 1997). In applying this strategy, recallresponses are detected by culturing PBL from subjects in vitro for 1-2weeks in the presence of a test peptide plus antigen presenting cells(APC) to allow activation of “memory” T cells, as compared to “naive” Tcells. At the end of the culture period, T cell activity is detectedusing assays for T cell activity including ⁵¹Cr release involvingpeptide-sensitized targets, T cell proliferation, or lymphokine release.

The following describes the peptide epitopes and corresponding nucleicacids of the invention in more detail.

Binding Affinity of Peptide Epitopes for HLA Molecules

As indicated herein, the large degree of HLA polymorphism is animportant factor to be taken into account with the epitope-basedapproach to vaccine development. To address this factor, epitopeselection encompassing identification of peptides capable of binding athigh or intermediate affinity to multiple HLA molecules is preferablyutilized, most preferably these epitopes bind at high or intermediateaffinity to two or more allele-specific HLA molecules.

CTL-inducing peptide epitopes of interest for vaccine compositionspreferably include those that have an IC₅₀ or binding affinity value fora class I HLA molecule(s) of 500 nM or better (i.e., the value is ≦500nM). HTL-inducing peptide epitopes preferably include those that have anIC₅₀ or binding affinity value for class II HLA molecules of 1000 nM orbetter, (i.e., the value is ≦1,000 nM). For example, peptide binding isassessed by testing the capacity of a candidate peptide to bind to apurified HLA molecule in vitro. Peptides exhibiting high or intermediateaffinity are then considered for further analysis. Selected peptides aregenerally tested on other members of the supertype family. In preferredembodiments, peptides that exhibit cross-reactive binding are then usedin cellular screening analyses or vaccines.

The relationship between binding affinity for HLA class I molecules andimmunogenicity of discrete peptide epitopes on bound antigens has beendetermined. As disclosed in greater detail herein, higher HLA bindingaffinity is correlated with greater immunogenicity.

Greater immunogenicity can be manifested in several different ways.Immunogenicity corresponds to whether an immune response is elicited atall, and to the vigor of any particular response, as well as to theextent of a population in which a response is elicited. For example, apeptide epitope might elicit an immune response in a diverse array ofthe population, yet in no instance produce a vigorous response. Inaccordance with these principles, close to 90% of high binding peptidehave been found to elicit a response and thus be “immunogenic,” ascontrasted with about 50% of the peptides that bind with intermediateaffinity. (See, e.g., Schaeffer et al. PNAS 1988) Moreover, not only didpeptides with higher binding affinity have an enhanced probability ofgenerating an immune response, the generated response tended to be morevigorous than the response seen with weaker binding peptides. As aresult, less peptide is required to elicit a similar biological effectif a high affinity binding peptide is used rather than a lower affinityone. Thus, in preferred embodiments of the invention, high affinitybinding epitopes are used.

The correlation between binding affinity and immunogenicity was analyzedby two different experimental approaches (see, e.g., Sette, et al., J.Immunol. 153:5586-5592, 1994)). In the first approach, theimmunogenicity of potential epitopes ranging in HLA binding affinityover a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. Inthe second approach, the antigenicity of approximately 100 differenthepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201binding motifs, was assessed by using PBL from acute hepatitis patients.Pursuant to these approaches, it was determined that an affinitythreshold value of approximately 500 nM (preferably 50 nM or less)determines the capacity of a peptide epitope to elicit a CTL response.These data are true for class I binding affinity measurements fornaturally processed peptide epitopes and for synthesized T cellepitopes. These data also indicate the important role of determinantselection in the shaping of T cell responses (see, e.g., Schaeffer etal. Proc. Natl. Acad. Sci. USA 86:4649-4653, 1989).

An affinity threshold associated with immunogenicity in the context ofHLA class II (i.e., HLA DR) molecules has also been delineated (see,e.g., Southwood et al. J. Immunology 160:3363-3373, 1998. In order todefine a biologically significant threshold of HLA class II bindingaffinity, a database of the binding affinities of 32 DR-restrictedepitopes for their restricting element (i.e., the HLA molecule thatbinds the epitope) was compiled. In approximately half of the cases (15of 32 epitopes), DR restriction was associated with high bindingaffinities, i.e. binding affinity values of 100 nM or less. In the otherhalf of the cases (16 of 32), DR restriction was associated withintermediate affinity (binding affinity values in the 1001000 nM range).In only one of 32 cases was DR restriction associated with an IC₅₀ of1000 nM or greater. Thus, 1000 nM is defined as an affinity thresholdassociated with immunogenicity in the context of DR molecules.

Vaccines of the present invention may also comprise epitopes that bindto MHC class II DR molecules. A greater degree of heterogeneity in bothsize and binding frame position of the motif, relative to the N and Ctermini of the peptide, exists for class II peptide ligands. Thisincreased heterogeneity of HLA class II peptide ligands is due to thestructure of the binding groove of the HLA class II molecule which,unlike its class I counterpart, is less physically constricted at bothends.

There are numerous additional supermotifs and motifs in addition to theA2 supermotif and the A2.1-allele specific motif. By inclusion of one ormore epitopes from other motifs or supermotifs, enhanced populationcoverage for major global ethnicities can be obtained.

Peptide Analogs

In general, CTL and HTL responses are not directed against all possibleepitopes. Rather, they are restricted to a few “immunodominant”determinants (Zinkernagel, et al., Adv. Immunol. 27:5159, 1979; Bennink,et al., J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol.146:3977-3984, 1991). It has been recognized that immunodominance(Benacerraf, et al., Science 175:273-279, 1972) could be explained byeither the ability of a given epitope to selectively bind a particularHLA protein (determinant selection theory) (Vitiello, et al., J.Immunol. 131:1635, 1983); Rosenthal, et al., Nature 267:156-158, 1977),or to be selectively recognized by the existing TCR (T cell receptor)specificities (repertoire theory) (Klein, J., IMMUNOLOGY, THE SCIENCE OFSELFNONSELF DISCRIMINATION, John Wiley & Sons, New York, pp. 270-310,1982). It has been demonstrated that additional factors, mostly linkedto processing events, can also play a key role in dictating, beyondstrict immunogenicity, which of the many potential determinants will bepresented as immunodominant (Sercarz, et al., Annu. Rev. Immunol.11:729-766, 1993).

The concept of dominance and subdominance is relevant to immunotherapyof both infectious diseases and malignancies. For example, in the courseof chronic viral disease, recruitment of subdominant epitopes can beimportant for successful clearance of the infection, especially ifdominant CTL or HTL specificities have been inactivated by functionaltolerance, suppression, mutation of viruses and other mechanisms(Franco, et al., Curr. Opin. Immunol. 7:524-531, 1995). In the case ofcancer and tumor antigens, CTLs recognizing at least some of the highestbinding affinity peptides might be functionally inactivated. Lowerbinding affinity peptides are preferentially recognized at these times,and may therefore be preferred in therapeutic or prophylacticanti-cancer vaccines.

In particular, it has been noted that a significant number of epitopesderived from known non-viral tumor associated antigens (TAA) bind HLAclass I with intermediate affinity (IC₅₀ in the 50-500 nM range) ratherthan at high affinity (IC₅₀ of less than 50 nM).

For example, it has been found that 8 of 15 known TAA peptidesrecognized by tumor infiltrating lymphocytes (TIL) or CTL bound in the50-500 nM range. (These data are in contrast with estimates that 90% ofknown viral antigens were bound by HLA class I molecules with IC₅₀ of 50nM or less, while only approximately 10% bound in the 50-500 nM range(Sette, et al., J. Immunol., 153:558-5592, 1994). In the cancer settingthis phenomenon is probably due to elimination or functional inhibitionof the CTL recognizing several of the highest binding peptides,presumably because of T cell tolerization events.

Without intending to be bound by theory, it is believed that because Tcells to dominant epitopes may have been clonally deleted, and selectingsubdominant epitopes may allow existing T cells to be recruited, whichwill then lead to a therapeutic or prophylactic response. However, thebinding of HLA molecules to subdominant epitopes is often less vigorousthan to dominant ones.

Accordingly, there is a need to be able to modulate the binding affinityof particular immunogenic epitopes for one or more HLA molecules, tothereby modulate the immune response elicited by the peptide, forexample to prepare analog peptides which elicit a more vigorousresponse. This ability to modulate both binding affinity and theresulting immune response in accordance with these principles greatlyenhances the usefulness of peptide epitope-based vaccines andtherapeutic agents.

Although peptides with suitable cross-reactivity among all alleles of asuperfamily are identified by the screening procedures described above,cross-reactivity is not always as complete as possible, and in certaincases procedures to increase cross-reactivity of peptides can be useful;moreover, such procedures can also be used to modify other properties ofthe peptides such as binding affinity or peptide stability. Havingestablished the general rules that govern cross-reactivity of peptidesfor HLA alleles within a given motif or supermotif, modification (i.e.,analoging) of the structure of peptides of particular interest in orderto achieve broader (or otherwise modified) HLA binding capacity can beperformed. More specifically, peptides that exhibit the broadestcross-reactivity patterns, can be produced in accordance with theteachings herein.

In brief, the analoging strategy utilizes the motifs or supermotifs thatcorrelate with binding to certain HLA molecules. Analog peptides can becreated by substituting amino acid residues at primary anchor, secondaryanchor, or at primary and secondary anchor positions. Generally, analogsare made for peptides that already bear a motif or supermotif. For anumber of the motifs or supermotifs, residues are defined which aredeleterious to binding to allele-specific HLA molecules or members ofHLA supertypes that bind the respective motif or supermotif.Accordingly, removal of such residues that are detrimental to bindingcan be performed. For example, in the case of the A3 supertype, when allpeptides that have such deleterious residues are removed from thepopulation of peptides used in the analysis, the incidence ofcross-reactivity increased from 22% to 37% (see, e.g., Sidney, J. etal., Hu. Immunol. 45:79, 1996). Examples of C35 peptide epitope analogsof the present invention are found in Table 4. In a particularlypreferred embodiment, the isolated polypeptides of the present inventioncomprise or, alternatively, consist of the following C35 peptide epitopeanalogs: for the peptide epitope G22 to C30 of SEQ ID NO:2 and FIG. 1B(i.e., GVRIVVEYC), the analog with either alanine or glycine substitutedfor cysteine at the ninth amino acid residue (i.e., GVRIVVEYA (SEQ IDNO: 161) or GVRIVVEYG (SEQ ID NO: 162)); for the peptide epitope I25 toC33 of SEQ ID NO:2 and FIG. 1B (i.e., IVVEYCEPC), the analog with eitheralanine or glycine substituted for the cysteine at the sixth amino acidresidue and/or the ninth amino acid residue (i.e., IVVEYAEPC (SEQ ID NO:163), IVVEYCEPA (SEQ ID NO: 164), IVVEYGEPC (SEQ ID NO: 165), IVVEYCEPG(SEQ ID NO: 166), IVVEYAEP A (SEQ ID NO: 167), IVVEYAEPG (SEQ ID NO:168), IVVEYGEPA (SEQ ID NO: 169), IVVEYGEPG (SEQ ID NO: 170)); for thepeptide epitope K77 to Y85 of SEQ ID NO: 2 and FIG. 1B (i.e.,KLENGGFPY), the analog with valine substituted for tyrosine at the ninthamino acid residue (i.e., KLENGGFPV (SEQ ID NO: 171)); for peptideepitope K104 to C112 of SEQ ID NO:2 and FIG. 1B (i.e., KITNSRPPC), theanalogs with alanine, glycine or leucine substituted for cysteine at theninth amino acid residue (i.e., KITNSRPPL (SEQ ID NO: 172), KITNSRPPA(SEQ ID NO: 173), KITNSRPPG (SEQ ID NO: 174)); for peptide epitope K104to V113 of SEQ ID NO:2 and FIG. 1B (i.e., KITNSRPPCV), the analogs withalanine, glycine, serine or leucine substituted for cysteine at theninth amino acid residue (i.e., KITNSRPPLV (SEQ ID NO: 175), KITNSRPPAV(SEQ ID NO: 176), KITNSRPPGV (SEQ ID NO: 177), KITNSRPPSV (SEQ ID NO:178)), for the peptide epitope I105 to V113 of SEQ ID NO:2 and FIG. 1B(i.e., ITNSRPPCV), the analogs wherein either leucine or methionine issubstituted for threonine at the second amino acid residue and/oralanine, serine or glycine is substituted for cysteine at the eighthamino acid residue (i.e., ILNSRPPCV (SEQ ID NO: 179), IMNSRPPCV (SEQ IDNO: 180), ITNSRPPAV (SEQ ID NO: 181), ITNSRPPGV (SEQ ID NO: 182),ILNSRPPAV (SEQ ID NO: 183), ILNSRPPGV (SEQ ID NO: 184), IMNSRPPAV (SEQID NO: 185), IMNSRPPGV (SEQ ID NO: 186), ILNSRPPSV (SEQ ID NO: 187),IMNSRPPSV (SEQ ID NO: 188), ITNSRPPSV (SEQ ID NO: 189)), for the peptideepitope N107 to L115 of SEQ ID NO:2 and FIG. 1B (i.e., NSRPPCVIL), theanalog with either alanine or glycine substituted for cysteine at thesixth amino acid residue (i.e., NSRPPAVIL (SEQ ID NO: 190), NSRPPGVIL(SEQ ID NO: 191)). The invention is further directed to polypeptidescomprising or, alternatively, consisting of one or more C35 epitopeanalogs. In a preferred embodiment, the invention is directed topolypeptides comprising one or more C35 epitope analogs and, inaddition, one or more C35 peptide epitopes. In a particularly preferredembodiment, the invention is directed to a fusion protein comprising atleast one C35 peptide epitope analog selected from the group consistingof: for the peptide epitope G22 to C30 of SEQ ID NO:2 and FIG. 1B (i.e.,GVRIVVEYC), the analog with either alanine or glycine substituted forcysteine at the ninth amino acid residue (i.e., GVRIVVEYA (SEQ ID NO:161) or GVRIVVEYG (SEQ ID NO: 162)); for the peptide epitope I25 to C33of SEQ ID NO:2 and FIG. 1B (i.e., IVVEYCEPC), the analog with eitheralanine or glycine substituted for the cysteine at the sixth amino acidresidue and/or the ninth amino acid residue (i.e., IVVEYAEPC (SEQ ID NO:163), IVVEYCEPA (SEQ ID NO: 164), IVVEYGEPC (SEQ ID NO: 165), IVVEYCEPG(SEQ ID NO: 166), IVVEYAEPA (SEQ ID NO: 167), IVVEYAEP G (SEQ ID NO:168), IVVEYGEPA (SEQ ID NO: 169), IVVEYGEPG (SEQ ID NO: 170)); for thepeptide epitope K77 to Y85 of SEQ ID NO: 2 and FIG. 1B (i.e.,KLENGGFPY), the analog with valine substituted for tyrosine at the ninthamino acid residue (i.e., KLENGGFPV (SEQ ID NO: 171)); for peptideepitope K104 to C112 of SEQ ID NO:2 and FIG. 1B (i.e., KITNSRPPC), theanalogs with alanine, glycine or leucine substituted for cysteine at theninth amino acid residue (i.e., KITNSRPPL (SEQ ID NO: 172), KITNSRPPA(SEQ ID NO: 173), KITNSRPPG (SEQ ID NO: 174)); for peptide epitope K104to V113 of SEQ ID NO:2 and FIG. 1B (i.e., KITNSRPPCV), the analogs withalanine, glycine, serine or leucine substituted for cysteine at theninth amino acid residue (i.e., KITNSRPPLV (SEQ ID NO: 175), KITNSRPPAV(SEQ ID NO: 176), KITNSRPPGV (SEQ ID NO: 177), KITNSRPPSV (SEQ ID NO:178)); for the peptide epitope I105 to V113 of SEQ ID NO:2 and FIG. 1B(i.e., ITNSRPPCV), the analogs wherein either leucine or methionine issubstituted for threonine at the second amino acid residue and/oralanine, serine or glycine is substituted for cysteine at the eighthamino acid residue (i.e., ILNSRPPCV (SEQ ID NO: 179), IMNSRPPCV (SEQ IDNO: 180), ITNSRPPAV (SEQ ID NO: 181), ITNSRPPGV (SEQ ID NO: 182),ILNSRPPAV (SEQ ID NO: 183), ILNSRPPGV (SEQ ID NO: 184), IMNSRPPAV (SEQID NO: 185), IMNSRPPGV (SEQ ID NO: 186), ILNSRPPSV (SEQ ID NO: 187), IMNSRPPSV (SEQ ID NO: 188), ITNSRPPSV (SEQ ID NO: 189)), for the peptideepitope N107 to L115 of SEQ ID NO:2 and FIG. 1B (i.e., NSRPPCVIL), theanalog with either alanine or glycine substituted for cysteine at thesixth amino acid residue (i.e., NSRPPAVIL (SEQ ID NO: 190), NSRPPGVIL(SEQ ID NO: 191)), and at least one C35 peptide epitope selected fromthe group consisting of: amino acids E4 to P12 of SEQ ID NO:2, S9 to V17of SEQ ID NO: 2, S21 to Y29 of SEQ ID NO:2, G22 to C30 of SEQ ID NO: 2,I25 to C33 of SEQ ID NO:2, T38 to V46 of SEQ ID NO:2, G61 to I69 of SEQID NO:2, T62 to N70 of SEQ ID NO:2, G63 to G71 of SEQ ID NO:2, F65 toL73 of SEQ ID NO: 2, I67 to F75 of SEQ ID NO:2, K77 to Y85 of SEQ IDNO:2, Q72 to E86 of SEQ ID NO:2, G81 to L89 of SEQ ID NO:2, G99 to V113of SEQ ID NO:2, E100 to V113 of SEQ ID NO:2, K104 to C112 of SEQ IDNO:2, K104 to V113 of SEQ ID NO: 2, I105 to V113 of SEQ ID NO:2, andN107 to L115 of SEQ ID NO:2.

Thus, one strategy to improve the cross-reactivity of peptides within agiven supermotif is simply to delete one or more of the deleteriousresidues present within a peptide and substitute a small “neutral”residue such as Ala (that may not influence T cell recognition of thepeptide). An enhanced likelihood of cross-reactivity is expected if,together with elimination of detrimental residues within a peptide,“preferred” residues associated with high affinity binding to anallele-specific HLA molecule or to multiple HLA molecules within asuperfamily are inserted.

To ensure that an analog peptide, when used as a vaccine, actuallyelicits a CTL response to the native epitope in vivo (or, in the case ofclass II epitopes, elicits helper T cells that cross-react with the wildtype peptides), the analog peptide may be used to induce T cells invitro from individuals of the appropriate HLA allele. Thereafter, theimmunized cells' capacity to lyse wild type peptide sensitized targetcells is evaluated. Alternatively, evaluation of the cells' activity canbe evaluated by monitoring IFN release. Each of these cell monitoringstrategies evaluate the recognition of the APC by the CTL. It will bedesirable to use as antigen presenting cells, cells that have beeneither infected, or transfected with the appropriate genes, or,(generally only for class II epitopes, due to the different peptideprocessing pathway for HLA class II), cells that have been pulsed withwhole protein antigens, to establish whether endogenously producedantigen is also recognized by the T cells induced by the analog peptide.It is to be noted that peptide/protein-pulsed dendritic cells can beused to present whole protein antigens for both HLA class I and classII.

Another embodiment of the invention is to create analogs of weak bindingpeptides, to thereby ensure adequate numbers of cellular binders. ClassI binding peptides exhibiting binding affinities of 500-5000 nM, andcarrying an acceptable but suboptimal primary anchor residue at one orboth positions can be “fixed” by substituting preferred anchor residuesin accordance with the respective supertype. The analog peptides canthen be tested for binding and/or cross-binding capacity.

Another embodiment of the invention is to create analogs of peptidesthat are already cross-reactive binders and are vaccine candidates, butwhich bind weakly to one or more alleles of a supertype. If thecross-reactive binder carries a suboptimal residue (less preferred ordeleterious) at a primary or secondary anchor position, the peptide canbe analoged by substituting out a deleterious residue and replacing itwith a preferred or less preferred one, or by substituting out a lesspreferred residue and replacing it with a preferred one. The analogpeptide can then be tested for cross-binding capacity.

Another embodiment for generating effective peptide analogs involves thesubstitution of residues that have an adverse impact on peptidestability or solubility in, e.g., a liquid environment. Thissubstitution may occur at any position of the peptide epitope. Forexample, a cysteine (C) can be substituted out in favor of α-aminobutyric acid. Due to its chemical nature, cysteine has the propensity toform disulfide bridges and sufficiently alter the peptide structurallyso as to reduce binding capacity. Substituting α-amino butyric acid forC not only alleviates this problem, but actually improves binding andcrossbinding capability in certain instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I.Chen, John Wiley & Sons, England, 1999). Substitution of cysteine withα-amino butyric acid may occur at any residue of a peptide epitope, i.e.at either anchor or non-anchor positions.

Moreover, it has been shown that in sets of A*0201 motif-bearingpeptides containing at least one preferred secondary anchor residuewhile avoiding the presence of any deleterious secondary anchorresidues, 69% of the peptides will bind A*0201 with an IC₅₀ less than500 nM (Ruppert, J. et al. Cell 74:929, 1993). The determination of whatwas a preferred or deleterious residue in Ruppert can be used togenerate algorithms (see, e.g., 22). Such algorithms are flexible inthat cut-off scores may be adjusted to select sets of peptides withgreater or lower predicted binding properties, as desired.

C35 epitopes containing cysteine residues have a tendency to dimerizewith other cysteine containing peptides. Thus, an embodiment of thepresent invention is a composition comprising a peptide epitope of theinvention (e.g. a C35 peptide epitope listed in any of Tables 1-3 or5-6, exclusive of E100 to R109 of SEQ ID NO:2) and a suitable reducingagent that protects the free sulfhydryl group of the cysteine residuebut does not otherwise inhibit epitope binding. In a preferredembodiment the composition comprises the peptide epitope ITNSRPPCV (I105to V113 of SEQ ID NO:2) or KITNSRPPCV (K104 to V113 of SEQ ID NO:2) incombination with a suitable reducing agent. Suitable reducing agentsinclude, but are not limited to, TCEP and dithiothreitol (DTT).

Another embodiment of the invention is to create peptide epitope analogsin which the cysteine residues of the peptide epitope (e.g., a C35peptide epitope listed in any of Tables 1-3 or 5-6, exclusive of E100 toR109 of SEQ ID NO:2) have been substituted with any other amino acid tofacilitate synthesis. (See Zarling, A. L. et al., J. Exp. Med. 192(12):1755-1762 (2000)). Preferably, the cysteine residues are substitutedwith either alanine, serine or glycine residues, although any amino acidcan be substituted provided that such substitution does not negativelyeffect binding to MHC or recognition by T cells. Thus, in a particularlypreferred embodiment, the isolated polypeptides of the present inventioncomprise or, alternatively, consist of the following C35 peptide epitopeanalogs: for the peptide epitope G22 to C30 of SEQ ID NO:2 and FIG. 1B(i.e., GVRIVVEYC), the analog with either alanine or glycine substitutedfor the cysteine at the ninth amino acid residue (i.e., GVRIVVEYA (SEQID NO: 161) or GVRIVVEYG (SEQ ID NO: 162)); for the peptide epitope I25to C33 of SEQ ID NO:2 and FIG. 1B (i.e., IVVEYCEPC), the analog witheither alanine or glycine substituted for the cysteine at the sixthamino acid residue and/or the ninth amino acid residue (i.e., IVVEYAEPC(SEQ ID NO: 163) or IVVEYGEPC (SEQ ID NO: 164) or IVVEYCEPA (SEQ ID NO:165) or IVVEYCEPG (SEQ ID NO: 166) or IVVEYAEPA (SEQ ID NO: 167) orIVVEYAEPG (SEQ ID NO: 168) or IVVEYGEPA (SEQ ID NO: 169) or IVVEYGEPG(SEQ ID NO: 170)); for the peptide epitope of K104 to C112 of SEQ IDNO:2 and FIG. 1B (i.e., KITNSRPPC) the analog with either alanine orglycine substituted for the cysteine at the ninth residue (i.e.,KITNSRPPA (SEQ ID NO: 171) or KITNSRPPG (SEQ ID NO: 172)); for thepeptide epitope K104 to V113 of SEQ ID NO:2 and FIG. 1B (i.e.,KITNSRPPCV), the analog with either alanine, serine or glycinesubstituted for the cysteine at the ninth residue (i.e., KITNSRPPAV (SEQID NO: 173), KITNSRPPSV (SEQ ID NO: 174) or KITNSRPPGV (SEQ ID NO:175)); for the peptide epitope I105 to V113 of SEQ ID NO:2 and FIG. 1B(i.e., ITNSRPPCV), the analog with either alanine, serine or glycinesubstituted for the cysteine at the eighth residue (i.e., ITNSRPPAV (SEQID NO: 176), ITNSRPPSV (SEQ ID NO: 177) or ITNSRPPGV (SEQ ID NO: 178));for the peptide epitope N107 to L115 (i.e., NSRPPCVIL), the analog witheither alanine or glycine substituted for the cysteine at the sixthamino acid residue (i.e., NSRPPAVIL (SEQ ID NO: 190), NSRPPGVIL (SEQ IDNO: 191)); for the multi-epitope peptide T101 to V113 of SEQ ID NO:2 andFIG. 1B (i.e., TLEKITNSRPPCV), the analog with either alanine or glycinesubstituted for the cysteine at the twelfth residue (i.e., TLEKITNSRPPAV(SEQ ID NO: 192) or TLEKITNSRPPGV (SEQ ID NO: 193)); for themulti-epitope peptide E100 to V113 of SEQ ID NO:2 and FIG. 1B (i.e.,ETLEKITNSRPPCV), the analog with either alanine or glycine substitutedfor the cysteine at the thirteenth amino acid residue (i.e.,ETLEKITNSRPPAV (SEQ ID NO: 194), ETLEKITNSRPPGV (SEQ ID NO: 195)), forthe multi-epitope peptide G99 to V113 of SEQ ID NO:2 and FIG. 1B (i.e.,GETLEKITNSRPPCV), the analog with either alanine or glycine substitutedfor the cysteine at the fourteenth amino acid residue (i.e.,GETLEKITNSRPPAV (SEQ ID NO: 196)), GETLEKITNSRPPGV (SEQ ID NO: 197)),for the multi-epitope peptide I93 to V113 of SEQ ID NO:2 and FIG. 1B(i.e., IRRASNGETLEKITNSRPPCV), the analog with either alanine or glycinesubstituted for the cysteine at the twentieth residue (i.e.,IRRASNGETLEKITNSRPPAV (SEQ ID NO: 198)) or IRRASNGETLEKITNSRPPGV (SEQ IDNO: 199)); for the multi-epitope peptide D88 to V113 of SEQ ID NO:2 andFIG. 1B (i.e., DLIEAIRRASNGETLEKITNSRPPCV), the analog with eitheralanine or glycine substituted for the cysteine at the twenty-fifthresidue (i.e., DLIEAIRRASNGETLEKITNSRPPAV (SEQ ID NO: 200) orDLIEAIRRASNGETLEKITNSRPPGV (SEQ ID NO: 201)); for the multi-epitopepeptide P84 to V113 of SEQ ID NO:2 and FIG. 1B (i.e.,PYEKDLIEAIRRASNGETLEKITNSRPPCV, the analog with either alanine orglycine substituted for the cysteine at the twenty-ninth residue (i.e.,PYEKDLIEAIRRASNGETLEKITNSRPPAV (SEQ ID NO: 202)) orPYEKDLIEAIRRASNGETLEKITNSRPPGV (SEQ ID NO: 203)); for the multi-epitopepeptide K77 to L115 of SEQ ID NO:2 and FIG. 1B (i.e.,KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV), the analog with either alanineor glycine substituted for the cysteine at the thirty-sixth residue(i.e., KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPAV (SEQ ID NO: 204)) orKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPGV (SEQ ID NO: 205)); for themulti-epitope peptide Q72 to L115 of SEQ ID NO:2 and FIG. 1B (i.e.,QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV), the analog with eitheralanine or glycine substituted for the cysteine at the forty-firstresidue (i.e., QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPAV (SEQ ID NO:206) or QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPGV (SEQ ID NO: 207));for the multi-epitope peptide F65 to L115 of SEQ ID NO:2 and FIG. 1B(i.e., FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV), the analogwith either alanine or glycine substituted for the cysteine at theforty-eighth residue (i.e.,FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPAV (SEQ ID NO: 208)) orFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPGV (SEQ ID NO: 209)); andfor the multi-epitope peptide L59 to L115 of SEQ ID NO:2 and FIG. 1B(i.e., LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNS RPPCV), theanalog with either alanine or glycine substituted for the cysteine atthe fifty-fourth residue (i.e.,LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNS RPPAV (SEQ ID NO:210)) or LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNS RPPGV (SEQID NO. 211)).

Another embodiment of the invention is to create peptide epitope analogsin which the cysteine residues of the peptide epitope (e.g., a C35peptide epitope listed in any of Tables 1-3 or 5-6, exclusive of E100 toR109 of SEQ ID NO:2, having one or more cysteine residues) have been“cysteinylated” (i.e., reacted with a second cysteine residue). (SeePierce, R. A. et al., J. Immunol. 163(12):6360-6364 (1999)). As usedherein, the term “cysteinylated” describes a cysteine residue, within apeptide (e.g. peptide epitope) of the present invention, which has beenreacted with a second free cysteine, i.e. a cysteine not part of alarger peptide, at the free sulfhydryl group thereby creating adisulfide bond (—SH+HS—=—S—S—).

Thus, in a particularly preferred embodiment, the isolated polypeptidesof the present invention comprise or, alternatively, consist of thefollowing C35 peptide epitope analogs: for the peptide epitope of K104to V113 of SEQ ID NO:2 and FIG. 1B (i.e., KITNSRPPCV), the analogwherein the cysteine at the ninth residue has been cysteinylated and forthe peptide epitope of I105 to V113 of SEQ ID NO:2 and FIG. 1B (i.e.ITNSRPPCV), the analog wherein the cysteine at the eighth residue hasbeen cysteinylated.

Another embodiment of the invention is to create peptide epitope analogsin which the serine, threonine and/or tyrosine residues of the peptideepitope (e.g., a C35 peptide epitope listed in any of Tables 1-3 or 5-6,exclusive of E100 to R109 of SEQ ID NO:2) have been phosphorylated.Thus, in a particularly preferred embodiment, the isolated polypeptidesof the present invention comprise or, alternatively, consist of thefollowing C35 peptide epitope analogs: for the peptide epitope E4 to P12of SEQ ID NO:2 and FIG. 1B (i.e., EPGQTSVAP), the analog wherein thethreonine at T8 and/or the serine at S9 have been phosphorylated; forthe peptide epitope S9 to V17 of SEQ ID NO:2 and FIG. 1B (i.e.,SVAPPPEEV), the analog wherein the serine at S9 has been phosphorylated;for the peptide epitope S21 to Y29 of SEQ ID NO:2 and FIG. 1B (i.e.,SGVRIVVEY), the analog wherein the serine at S21 and/or the tyrosine atY29 are phosphorylated; for the peptide epitope G22 to C30 of SEQ IDNO:2 and FIG. 1B (i.e., GVRIVVEYC), the analog wherein the tyrosine atY29 is phosphorylated; for the peptide epitope T38 to V46 of SEQ ID NO:2and FIG. 1B (i.e., TYLELASAV), the analog wherein the threonine at T38,the tyrosine at Y39, and/or the serine at S44 are phosphorylated; forthe peptide epitope G61 to I69 (i.e., GTGAFEIEI), the analog wherein thethreonine at T62 is phosphorylated); for the peptide epitope T62 to N70of SEQ ID NO:2 and FIG. 1B (i.e., TGAFEIEIN), the analog wherein thethreonine at T62 has been phosphorylated; for the peptide epitope K77 toY85 of SEQ ID NO:2 and FIG. 1B (i.e., KLENGGFPY), the analog wherein thetyrosine at Y85 is phosphorylated; for the peptide epitope Q72 to E86 ofSEQ ID NO:2 and FIG. 1B (i.e., QLVFSKLENGGFPYE), the analog wherein theserine at S76 and/or the tyrosine at Y85 are phosphorylated; for thepeptide epitope G81 to L89 of SEQ ID NO:2 or FIG. 1B (i.e., GGFPYEKDL),the analog wherein the tyrosine at Y85 is phosphorylated; for thepeptide epitope K104 to C112 of SEQ ID NO:2 and FIG. 1B (i.e.,KITNSRPPC), the analog wherein the threonine at T106 and/or the serineat S108 are phosphorylated; for the peptide epitope K104 to V113 of SEQID NO:2 and FIG. 1B (i.e., KITNSRPPCV), the analog wherein the threonineat T106 and/or the serine at S108 are phosphorylated; for the peptideepitope I105 to V113 (i.e., ITNSRPPCV), the analog wherein the threonineat T106 and/or the serine at S108 are phosphorylated; for the peptideepitope N107 to L115 (i.e., NSRPPCVIL), the analog wherein the serine atS108 is phosphorylated; for the polyepitopic peptide T101 to V113 of SEQID NO:2 and FIG. 1B (i.e., TLEKITNSRPPCV), the analog wherein thethreonines at T101 and T106 and/or the serine at S108 arephosphorylated; for the polyepitopic peptide I93 to V113 of SEQ ID NO:2and FIG. 1B (i.e., IRRASNGETLEKITNSRPPCV), the analog wherein the serineat S97 and/or the threonine at T101 and/or the threonine at T106 and/orthe serine at S108 are phosphorylated; for the polyepitopic peptide D88to V113 of SEQ ID NO:2 and FIG. 1B (i.e., DLIEAIRRASNGETLEKITNSRPPCV),the analog wherein the serine at S97 and/or the threonine at T101 and/orthe threonine at T106 and/or the serine at S108 are phosphorylated; forthe polyepitopic peptide P84 to V113 of SEQ ID NO:2 and FIG. 1B (i.e.,PYEKDLIEAIRRASNGETLEKITNSRPPCV), the analog wherein the tyrosine at Y85and/or the serine at S97 and/or the threonine at T101 and/or thethreonine at T106 and/or the serine at S108 are phosphorylated; for thepolyepitopic peptide K77 to V113 of SEQ ID NO:2 and FIG. 1B (i.e.,KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV), the analog wherein the tyrosineat Y85 and/or the serine at S97 and/or the threonine at T101 and/or thethreonine at T106 and/or the serine at S108 are phosphorylated; for thepolyepitopic peptide Q72 to V113 of SEQ ID NO:2 and FIG. 1B (i.e.,QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV), the analog wherein theserine at S76 and/or the tyrosine at Y85 and/or the serine at S97 and/orthe threonine at T101 and/or the threonine at T106 and/or the serine atS108 are phosphorylated; for the polyepitopic peptide F65 to V113 of SEQID NO:2 and FIG. 1B (i.e.,FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV), the analog whereinthe serine at S76 and/or the tyrosine at Y85 and/or the serine at S97and/or the threonine at T101 and/or the threonine at T106 and/or theserine at S108 are phosphorylated; for the polyepitopic peptide L59 toV113 of SEQ ID NO:2 and FIG. 1B (i.e.,LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNS RPPCV), the analogwherein the threonine at T62 and/or the serine at S76 and/or thetyrosine at Y85 and/or the serine at S97 and/or the threonine at T101and/or the threonine at T106 and/or the serine at S108 arephosphorylated.

Another embodiment of the invention is to create peptide epitope analogsin which the asparagine residues of the peptide epitope (e.g., a C35peptide epitope listed in any of Tables 1-3 or 5-6, exclusive of E100 toR109 of SEQ ID NO:2) have been converted to aspartic acid aftertranslation. (See Skipper, J. C. et al., J. Exp. Med. 183(2):527-534(1996)).

In preferred embodiments, the C35 peptide epitope analogs of the presentinvention contain multiple modifications provided that suchmodifications do not inhibit binding to MHC molecules or recognition byT cells. Thus, preferred analogs include C35 peptide epitopes for whichone or more residues have been modified as described herein to increasebinding affinity to MHC molecules, one or more cysteine residues havebeen replaced with alanine or glycine residues to facilitate synthesis,and one or more serine, threonine or tyrosine residues have beenphosphorylated.

Furthermore, additional amino acids can be added to the termini of apeptide epitope to provide for ease of linking peptide epitopes one toanother, for coupling to a carrier support or larger polypeptide, formodifying the physical or chemical properties of the peptide oroligopeptide, or the like. Amino acids such as tyrosine, cysteine,lysine, glutamic or aspartic acid, or the like, can be introduced at theC- or N-terminus of the peptide or oligopeptide, particularly class Ipeptides. It is to be noted that modification at the carboxyl terminusof a CTL epitope may, in some cases, alter binding characteristics ofthe peptide. In addition, the peptide or oligopeptide sequences candiffer from the natural sequence by being modified by terminal-NH₂acylation, e.g., by alkanoyl (C₁-C₂₀) or thioglycolyl acetylation,terminal-carboxylamidation, e.g., ammonia, methylamine, etc.,polyethylene-glycol modification (i.e., PEGylation) of the C-terminus,and the addition of a lipid tail (e.g., a palmitoyl-lysine chain) toenhance presentation to T cells and immunogenicity. (See Brinckerhoff,L. H. et al., Int. J. Cancer 83(3):326-334 (1999); Le Gal, F. A. et al.,Int. J. Cancer 98(2):221-227 (2002). N-terminal amides, in particular,will be more resistant to certain peptidases, thus preventingdestruction of the peptide epitope in situ without affectingrecognition. This will effectively increase the half-life of the peptideepitope and enhance its ability to stimulate immune cells. In someinstances these modifications may provide sites for linking to a supportor other molecule.

Preparation of Peptide Epitopes

Peptide epitopes in accordance with the invention can be preparedsynthetically, by recombinant DNA technology or chemical synthesis, orfrom natural sources such as native tumors or pathogenic organisms.Peptide epitopes may be synthesized individually or as polyepitopicpolypeptides (e.g., homopolymers or heteropolymers). Although thepeptide will preferably be substantially free of other naturallyoccurring host cell proteins and fragments thereof, in some embodimentsthe peptides may be synthetically conjugated to native fragments orparticles.

In addition, one or more non-C35 tumor associated peptides can be linkedto one or more C35 peptide epitopes and/or C35 peptide epitope analogsto increase immune response via HLA class I and/or class II. Especiallypreferred are polypeptides comprising a series of epitopes, known as“polytopes,” and nucleic acids encoding same. The epitopes can bearranged in sequential or overlapping fashion (see, e.g., Thomson etal., Proc. Natl. Acad. Sci. USA 92:5845-5849 (1995); Gilbert et al.,Nature/Biotechnology 15:1280-1284 (1997)), with or without the naturalflanking sequences, and can be separated by unrelated linker sequencesif desired. The polytope is processed to generate individual epitopeswhich are recognized by the immune system for generation of immuneresponses.

Thus, for example, C35 peptide epitopes and C35 peptide epitope analogscan be combined with peptides from other tumor rejection antigens (e.g.,by preparation of hybrid nucleic acids or polypeptides) to form“polytopes.” (Zeng, G. et al., Proc. Natl. Acad. Sci. 98(7):3964-3969(2001); Zeng, G. et al., J. Immunol. 165:1153-1159 (2000); Mancini, S.et al., J. Exp. Med. 189(5):871-876 (1999)). Exemplary tumor associatedantigens that can be administered to induce or enhance an immuneresponse are derived from tumor associated genes and encoded proteinsincluding: MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, MAGE-7,MAGE-8, MAGE-9, MAGE-10, MAGE-11, MAGE-12, MAGE-13, GAGE-1, GAGE-2,GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1,LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4(MAGE-B4), tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE-C1,MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4,SSX-5, SCP-1 and CT-7. For example, specific antigenic peptidescharacteristic of tumors include those listed in Table A.

TABLE A Gene MHC Peptide Position SEQ ID NO: MAGE-1 HLA-A1 EADPTGHSY161-169 212 HLA-Cw16 SAYGEPRKL 230-238 213 MAGE-3 HLA-A1 EVDPIGHLY168-176 214 HLA-A2 FLWGPRALV 271-279 215 HLA-B44 MEVDPIGHLY 167-176 216BAGE HLA-Cw16 AARAVFLAL  2-10 217 GAGE-1,2 HLA-Cw16 YRPRPRRY  9-16 218RAGE HLA-B7 SPSSNRIRNT 11-20 219 GnT-V HLA-A2 VLPDVFIRC(V)    2-10/11220 MUM-1 HLA-B44 EEKLIVVLF exon 2/intron 221 EEKLSVVLF (wild-type) 222CDK4 HLA-A2 ACDPHSGHFV 23-32 223 ARDPHSGHFV (wild-type) 224 β-cateninHLA-A24 SYLDSGIHF 29-37 225 SYLDSGIHS (wild-type) 226 Tyrosinase HLA-A2MLLAVLYCL 1-9 227 HLA-A2 YMNGTMSQV 369-377 228 HLA-A2 YMDGTMSQV 369-377229 HLA-A24 AFLPWHRLF 206-214 230 HLA-B44 SEIWRDIDF 192-200 231 HLA-B44YEIWRDIDF 192-200 232 HLA-DR4 QNILLSNAPLGPQFP 56-70 233 HLA-DR4DYSYLQDSDPDSFQD 448-462 234 Melan-A^(Mart1) HLA-A2 (E)AAGIGILTV26/27-35   235 HLA-A2 ILTVILGVL 32-40 236 gp100^(Pme117) HLA-A2KTWGQYWQV 154-162 237 HLA-A2 ITDQVPFSV 209-217 238 HLA-A2 YLEPGPVTA280-288 239 HLA-A2 LLDGTATLRL 457-466 240 HLA-A2 VLYRYGSFSV 476-485 241PRAME HLA-A24 LYVDSLFFL 301-309 242 MAGE-6 HLA-Cw16 KISGGPRISYPL 292-303243 NY-ESO-1 HLA-A2 SLLMWITQCFL 157-167 244 HLA-A2 SLLMWITQC 157-165 245HLA-A2 QLSLLMWIT 155-163 246

Other examples of non-C35 HLA class I and HLA class II binding peptideswill be known to one of ordinary skill in the art and can be used in theinvention in a like manner to those disclosed herein. One of ordinaryskill in the art can prepare polypeptides comprising one or more C35peptide epitopes or C35 peptide epitope analogs and one or more of theaforementioned tumor rejection peptides, or nucleic acids encoding suchpolypeptides, according to standard procedures in molecular biology.Examples of polytopes comprising C35 peptide epitopes or C35 peptideepitope analogs of the present invention and various tumor rejectionantigenic peptides are set forth in Tables B and C below.

Thus, polytopes are groups of two or more potentially immunogenic orimmune response stimulating peptides which can be joined together invarious arrangements (e.g. concatenated, overlapping). The polytope (ornucleic acid encoding the polytope) can be administered in a standardimmunization protocol, e.g. to animals, to test the effectiveness of thepolytope in stimulating, enhancing and/or provoking an immune response.The peptides can be joined directly or via the use of flanking sequencesto form polytopes, and the use of polytopes as vaccines is well known inthe art.

In a preferred embodiment, the isolated polypeptides of the presentinvention comprise one or more C35 peptide epitopes or C35 peptideepitope analogs linked to one or more tumor rejection peptides. In aparticularly preferred embodiment, said one or more C35 peptide epitopesare selected from the group consisting of: amino acids E4 to P12 of SEQID NO:2, amino acids S9 to V17 of SEQ ID NO:2, amino acids S21 to Y29 ofSEQ ID NO:2, amino acids G22 to C30 of SEQ ID NO: 2, amino acids I25 toC33 of SEQ ID NO:2, amino acids T38 to V46 of SEQ ID NO:2, amino acidsG61 to I69 of SEQ ID NO:2, amino acids T62 to N70 of SEQ ID NO:2, aminoacids G63 to G71 of SEQ ID NO:2, amino acids F65 to L73 of SEQ ID NO:2,amino acids I67 to F75 of SEQ ID NO:2, amino acids K77 to Y85 of SEQ IDNO:2, amino acids Q72 to E86 of SEQ ID NO:2, amino acids G81 to L89 ofSEQ ID NO:2, amino acids K104 to C112 of SEQ ID NO:2, amino acids K104to V113 of SEQ ID NO:2, amino acids I105 to V113 of SEQ ID NO:2, aminoacids N107 to L115 of SEQ ID NO:2, amino acids T101 to V113 of SEQ IDNO:2, amino acids E100 to V113 of SEQ ID NO:2, amino acids G99 to V113of SEQ ID NO:2, amino acids I93 to V113 of SEQ ID NO:2, amino acids D88to V113 of SEQ ID NO:2, amino acids P84 to V113 of SEQ ID NO:2, aminoacids K77 to V113 of SEQ ID NO:2, amino acids Q72 to V113 of SEQ IDNO:2, amino acids F65 to V113 of SEQ ID NO:2, and L57 to V113 of SEQ IDNO:2; and said one or more tumor rejection peptides are selected fromthe group consisting of the antigenic peptides shown in Table A.

In another embodiment, one or more non-C35 cell penetrating peptides canbe linked to one or more C35 peptide epitopes and/or C35 peptide epitopeanalogs to enhance delivery of C35 peptide epitopes to cells, e.g.,dendritic cells. Especially preferred are polypeptides comprising aseries of C35 peptide epitopes or C35 peptide epitope analogs and cellpenetrating peptides, and nucleic acids encoding same. The epitopes andpeptides can be arranged in sequential or overlapping fashion with orwithout the natural flanking sequences, and can be separated byunrelated linker sequences if desired. The polypeptide is processed togenerate individual C35 epitopes which are recognized by the immunesystem for generation of immune responses.

Thus, for example, C35 peptide epitopes and C35 peptide epitope analogscan be combined with cell-penetrating peptides. (Wang, R.-F. et al.,Nature Biotechnology 20(2):149-154 (2002); Frankel, A. D. et al., Cell55:1189-1193 (1988); Elliott, G. et al., Cell 88(2):223-233 (1997);Phelan, A. et al., Nature Biotechnology 16(5):440-443 (1998); Lin, Y.-Z.et al., J. Biol. Chem. 270(24):14255-14258 (1995); Rojas, M. et al.,Nature Biotechnology 16(4):370-375 (1998)). Exemplary cell penetratingpeptides that can be administered to enhance delivery of C35 peptides tocells, such as dendritic cells, include: the Tat protein of humanimmunodeficiency virus, the HSV-1 structural protein VP22, and the12-residue membrane-translocating sequence (MTS) modified from the16-residue h region of the signal sequence of Kaposi fibroblast growthfactor.

The one or more C35 peptide epitopes/analogs and one or more cellpenetrating peptides can be joined together in various arrangements(e.g. concatenated, overlapping). The resulting polypeptide (or nucleicacid encoding the polypeptide) can be administered in a standardimmunization protocol, e.g. to animals, to test the effectiveness of thepolypeptide in stimulating, enhancing and/or provoking an immuneresponse. The C35 peptide epitopes/analogs and one or more cellpenetrating peptides can be joined directly or via the use of flankingsequences to form the polypeptides, and the use of such polypeptides asvaccines is well known in the art. Examples of polypeptides comprisingC35 peptide epitopes or C35 peptide epitope analogs of the presentinvention and various cell penetrating peptides are set forth in TablesD and E below.

TABLE B C35 Peptide/Epitope Exemplary Tumor Rejection Peptide ExemplaryPolytopes S9-V17 of SEQ ID NO: 2 amino acids 161-169 of MAGE-1SVAPPPEEVEADPTGHSY (SEQ ID NO: 247), SVAPPPEEVEADPTGHSYSVAAPPPEEVEADPTGHSY (SEQ ID NO: 248) amino acids 230-238 ofMAGE-1 SVAPPPEEVSAYGEPRKL (SEQ ID NO: 249), SAYGEPRKL SVAPPPEEVSAYGEPRKL(SEQ ID NO: 250) amino acids 168-176 of MAGE-3 SVAPPPEEVEVDPIGHLY (SEQID NO: 251), EVDPIGHLYSVAPPPEEVEVDPIGHLY (SEQ ID NO: 252) amino acids271-279 of MAGE-3 SVAPPPEEVFLWGPRALV (SEQ ID NO: 253),FLWGPRALVSVAPPPEEVFLWGPRALV (SEQ ID NO: 254) amino acids 167-176 ofMAGE-3 SVAPPPEEVMEVDPIGHLY (SEQ ID NO: 255),MEVDPIGHLYSVAPPPEEVMEVDPIGHLY (SEQ ID NO: 256) amino acids 2-10 of BAGESVAPPPEEVAARAVFLAL (SEQ ID NO: 257), AARAVFLALSVAPPPEEVAARAVFLAL (SEQ IDNO: 258) amino acids 9-16 of GAGE-1,2 SVAPPPEEVYRPRPRRY (SEQ ID NO:259), YRPRPRRYSVAPPPEEVYRPRPRRY (SEQ ID NO: 260) amino acids 11-20 ofRAGE SVAPPPEEVSPSSNRIRNT (SEQ ID NO: 261), SPSSNRIRNTSVAPPPEEVSPSSNRIRNT(SEQ ID NO: 262) amino acids 23-32 of CDK4 SVAPPPEEVARDPHSGHFV (SEQ IDNO: 263), ARDPHSGHFVSVAPPPEEVARDPHSGHFV (SEQ ID NO: 264) amino acids29-37 of β-catenin SVAPPPEEVSYLDSGIHS (SEQ ID NO: 265),SYLDSGIHSSVAPPPEEVSYLDSGIHS (SEQ ID NO: 266) amino acids 1-9 ofTyrosinase SVAPPPEEVMLLAVLYCL (SEQ ID NO: 267),MLLAVLYCLSVAPPPEEVMLLAVLYCL (SEQ ID NO: 268) amino acids 206-214 ofTyrosinase SVAPPPEEVAFLPWHRLF (SEQ ID NO: 269),AFLPWHRLFSVAPPPEEVAFLPWHRLF (SEQ ID NO: 270) amino acids 56-70 ofTyrosinase SVAPPPEEVQNILLSNAPLGPQFP (SEQ ID NO: 271),QNILLSNAPLGPQFPSVAPPPEEVQNILLSNAPLGPQFP (SEQ ID NO: 272) amino acids448-462 of Tyrosinase SVAPPPEEVDYSYLQDSDPDSFQD (SEQ ID NO: 273),DYSYLQDSDPDSFQDSVAPPPEEVDYSYLQDSDPDSFQD (SEQ ID NO: 274) amino acids32-40 of Melan-A^(MART-1) SVAPPPEEVJLTVILGVL (SEQ ID NO: 275),JLTVILGVLSVAPPPEEVJLTVILGVL (SEQ ID NO: 276) amino acids 154-162 ofgp100^(Pme117) SVAPPPEEVKTWGQYWQV (SEQ ID NO: 277),KTWGQYWQVSVAPPPEEVKTWGQYWQV (SEQ ID NO: 278) amino acids 209-217 ofgp100^(Pme117) SVAPPPEEVITDQVPFSV (SEQ ID NO: 279),ITDQVPFSVSVAPPPEEVITDQVPFSV (SEQ ID NO: 280) amino acids 280-288 ofgp100^(Pme117) SVAPPPEEVYLEPGPVTA (SEQ ID NO: 281),YLEPGPVTASVAPPPEEVYLEPGPVTA (SEQ ID NO: 282) amino acids 457-466 ofgp100^(Pme117) SVAPPPEEVLLDGTATLRL (SEQ ID NO: 283),LLDGTATLRLSVAPPPEEVLLDGTATLRL (SEQ ID NO: 284) amino acids 476-485 ofgp100^(Pme117) SVAPPPEEVVLYRYGSFSV (SEQ ID NO: 285),VLYRYGSFSVSVAPPPEEVVLYRYGSFSV (SEQ ID NO: 286) amino acids 301-309 ofPRAME SVAPPPEEVLYVDSLFFL (SEQ ID NO: 287), LYVDSLFFLSVAPPPEEVLYVDSLFFL(SEQ ID NO: 288) amino acids 292-303 of MAGE-6 SVAPPPEEVKISGGPRISYPL(SEQ ID NO: 289), KISGGPRISYPLSVAPPPEEVKISGGPRISYPL (SEQ ID NO: 290)amino acids 157-167 of NY-ESO-1 SVAPPPEEVSLLMWITQCFL (SEQ ID NO: 291),SLLMWITQCFLSVAPPPEEVSLLMWITQCFL (SEQ ID NO: 292) amino acids 157-165 ofNY-ESO-1 SVAPPPEEVSLLMWITQC (SEQ ID NO: 293),SLLMWITQCSVAPPPEEVSLLMWITQC (SEQ ID NO: 294) amino acids 155-163 ofNY-ESO-1 SVAPPPEEVQLSLLMWIT (SEQ ID NO: 295),QLSLLMWITSVAPPPEEVQLSLLMWIT (SEQ ID NO: 296) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFSVAPPPEEVTSYVKVLHHMVKISG (SEQ ID NO: 297)amino acids 281-295 of MAGE-3 S21-Y29 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 SGVRIVVEYEADPTGHSY (SEQ ID NO: 298), SGVRIVVEYEADPTGHSYSGVRIVVEYEADPTGHSY (SEQ ID NO: 299) amino acids 230-238 ofMAGE-1 SGVRIVVEYSAYGEPRKL (SEQ ID NO: 300), SAYGEPRKLSGVRIVVEYSAYGEPRKL(SEQ ID NO: 301) amino acids 168-176 of MAGE-3 SGVRIVVEYEVDPIGHLY (SEQID NO: 302), EVDPIGHLYSGVRIVVEYEVDPIGHLY (SEQ ID NO: 303) amino acids271-279 of MAGE-3 SGVRIVVEYFLWGPRALV (SEQ ID NO: 304),FLWGPRALVSGVRIVVEYFLWGPRALV (SEQ ID NO: 305) amino acids 167-176 ofMAGE-3 SGVRIVVEYMEVDPIGHLY (SEQ ID NO: 306),MEVDPIGHLYSGVRIVVEYMEVDPIGHLY (SEQ ID NO: 307) amino acids 2-10 of BAGESGVRIVVEYAARAVFLAL (SEQ ID NO: 308), AARAVFLALSGVRIVVEYAARAVFLAL (SEQ IDNO: 309) amino acids 9-16 of GAGE-1,2 SGVRIVVEYYRPRPRRY (SEQ ID NO:310), YRPRPRRYSGVRIVVEYYRPRPRRY (SEQ ID NO: 311) amino acids 11-20 ofRAGE SGVRIVVEYSPSSNRIRNT (SEQ ID NO: 312), SPSSNRIRNTSGVRIVVEYSPSSNRIRNT(SEQ ID NO: 313) amino acids 23-32 of CDK4 SGVRIVVEYACDPHSGHFV (SEQ IDNO: 314), ACDPHSGHFVSGVRIVVEYACDPHSGHFV (SEQ ID NO: 315) amino acids29-37 of β-catenin SGVRIVVEYSYLDSGIHF (SEQ ID NO: 316),SYLDSGIHFSGVRIVVEYSYLDSGIHF (SEQ ID NO: 317) amino acids 1-9 oftyrosinase SGVRIVVEYMLLAVLYCL (SEQ ID NO: 318),MLLAVLYCLSGVRIVVEYMLLAVLYCL (SEQ ID NO: 319) amino acids 206-214 oftyrosinase SGVRIVVEYAFLPWHRLF (SEQ ID NO: 320),AFLPWHRLFSGVRIVVEYAFLPWHRLF (SEQ ID NO: 321) amino acids 56-70 oftyrosinase SGVRIVVEYQNILLSNAPLGPQFP (SEQ ID NO: 322),QNILLSNAPLGPQFPSGVRIVVEYQNILLSNAPLGPQFP (SEQ ID NO: 323) amino acids448-462 of tyrosinase SGVRIVVEYDYSYLQDSDPDSFQD (SEQ ID NO: 324),DYSYLQDSDPDSFQDSGVRIVVEYDYSYLQDSDPDSFQD (SEQ ID NO: 325) amino acids32-40 of Melan-A^(MART-1) SGVRIVVEYJLTVILGVL (SEQ ID NO: 326),JLTVILGVLSGVRIVVEYJLTVILGVL (SEQ ID NO: 327) amino acids 154-162 ofgp100^(Pme117) SGVRIVVEYKTWGQYWQV (SEQ ID NO: 328),KTWGQYWQVSGVRIVVEYKTWGQYWQV (SEQ ID NO: 329) amino acids 209-217 ofgp100^(Pme117) SGVRIVVEY ITDQVPFSV (SEQ ID NO: 330), ITDQVPFSV SGVRIVVEYITDQVPFSV (SEQ ID NO: 331) amino acids 280-288 of gp100^(Pme117)SGVRIVVEYYLEPGPVTA (SEQ ID NO: 332), YLEPGPVTASGVRIVVEYYLEPGPVTA (SEQ IDNO: 333) amino acids 457-466 of gp100^(Pme117) SGVRIVVEYLLDGTATLRL (SEQID NO: 334), LLDGTATLRLSGVRIVVEYLLDGTATLRL (SEQ ID NO: 335) amino acids476-485 of gp100^(Pme117) SGVRIVVEYVLYRYGSFSV (SEQ ID NO: 336),VLYRYGSFSVSGVRIVVEYVLYRYGSFSV (SEQ ID NO: 337) amino acids 301-309 ofPRAME SGVRIVVEYLYVDSLFFL (SEQ ID NO: 338), LYVDSLFFLSGVRIVVEYLYVDSLFFL(SEQ ID NO: 339) amino acids 292-303 of MAGE-6 SGVRIVVEYKISGGPRISYPL(SEQ ID NO: 340), KISGGPRISYPLSGVRIVVEYKISGGPRISYPL (SEQ ID NO: 341)amino acids 157-167 of NY-ESO-1 SGVRIVVEYSLLMWITQCFL (SEQ ID NO: 342),SLLMWITQCFLSGVRIVVEYSLLMWITQCFL (SEQ ID NO: 343) amino acids 157-165 ofNY-ESO-1 SGVRIVVEYSLLMWITQC (SEQ ID NO: 344),SLLMWITQCSGVRIVVEYSLLMWITQC (SEQ ID NO: 345) amino acids 155-163 ofNY-ESO-1 SGVRIVVEYQLSLLMWIT (SEQ ID NO: 346),QLSLLMWITSGVRIVVEYQLSLLMWIT (SEQ ID NO: 347) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFSGVRIVVEYTSYVKVLHHMVKISG (SEQ ID NO: 348)amino acids 281-295 of MAGE-3 G22-C30 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 GVRIVVEYCEADPTGHSY (SEQ ID NO: 349), GVRIVVEYCEADPTGHSYGVRIVVEYCEADPTGHSY (SEQ ID NO: 350) amino acids 230-238 ofMAGE-1 GVRIVVEYCSAYGEPRKL (SEQ ID NO: 351), SAYGEPRKLGVRIVVEYCSAYGEPRKL(SEQ ID NO: 352) amino acids 168-176 of MAGE-3 GVRIVVEYCEVDPIGHLY (SEQID NO: 353), EVDPIGHLYGVRIVVEYCEVDPIGHLY (SEQ ID NO: 354) amino acids271-279 of MAGE-3 GVRIVVEYCFLWGPRALV (SEQ ID NO: 355),FLWGPRALVGVRIVVEYCFLWGPRALV (SEQ ID NO: 356) amino acids 167-176 ofMAGE-3 GVRIVVEYCMEVDPIGHLY (SEQ ID NO: 357),MEVDPIGHLYGVRIVVEYCMEVDPIGHLY (SEQ ID NO: 358) amino acids 2-10 of BAGEGVRIVVEYCAARAVFLAL (SEQ ID NO: 359), AARAVFLALGVRIVVEYCAARAVFLAL (SEQ IDNO: 360) amino acids 9-16 of GAGE-1,2 GVRIVVEYCYRPRPRRY (SEQ ID NO:361), YRPRPRRYGVRIVVEYCYRPRPRRY (SEQ ID NO: 362) amino acids 11-20 ofRAGE GVRIVVEYCSPSSNRIRNT (SEQ ID NO: 363), SPSSNRIRNTGVRIVVEYCSPSSNRIRNT(SEQ ID NO: 364) amino acids 23-32 of CDK4 GVRIVVEYCACDPHSGHFV (SEQ IDNO: 365), ACDPHSGHFVGVRIVVEYCACDPHSGHFV (SEQ ID NO: 366) amino acids29-37 of β-catenin GVRIVVEYCSYLDSGIHF (SEQ ID NO: 367),SYLDSGIHFGVRIVVEYCSYLDSGIHF (SEQ ID NO: 368) amino acids 1-9 oftyrosinase GVRIVVEYCMLLAVLYCL (SEQ ID NO: 369),MLLAVLYCLGVRIVVEYCMLLAVLYCL (SEQ ID NO: 370) amino acids 206-214 oftyrosinase GVRIVVEYCAFLPWHRLF (SEQ ID NO: 371),AFLPWHRLFGVRIVVEYCAFLPWHRLF (SEQ ID NO: 372) amino acids 56-70 oftyrosinase GVRIVVEYCQNILLSNAPLGPQFP (SEQ ID NO: 373),QNILLSNAPLGPQFPGVRIVVEYCQNILLSNAPLGPQFP (SEQ ID NO: 374) amino acids448-462 of tyrosinase GVRIVVEYCDYSYL QDSDPDSFQD (SEQ ID NO: 375),DYSYLQDSDPDSFQDGVRIVVEYCDYSYLQDSDPDSFQD (SEQ ID NO: 376) amino acids32-40 of Melan-A^(MART-1) GVRIVVEYCJLTVILGVL (SEQ ID NO: 377),JLTVILGVLGVRIVVEYCJLTVILGVL (SEQ ID NO: 378) amino acids 154-162 ofgp100^(Pme117) GVRIVVEYCKTWGQYWQV (SEQ ID NO: 379),KTWGQYWQVGVRIVVEYCKTWGQYWQV (SEQ ID NO: 380) amino acids 209-217 ofgp100^(Pme117) GVRIVVEYCITDQVPFSV (SEQ ID NO: 381),ITDQVPFSVGVRIVVEYCITDQVPFSV (SEQ ID NO: 382) amino acids 280-288 ofgp100^(Pme117) GVRIVVEYCYLEPGPVTA (SEQ ID NO: 383),YLEPGPVTAGVRIVVEYCYLEPGPVTA (SEQ ID NO: 384) amino acids 457-466 ofgp100^(Pme117) GVRIVVEYCLLDGTATLRL (SEQ ID NO: 385),LLDGTATLRLGVRIVVEYCLLDGTATLRL (SEQ ID NO: 386) amino acids 476-485 ofgp100^(Pme117) GVRIVVEYCVLYRYGSFSV (SEQ ID NO: 387),VLYRYGSFSVGVRIVVEYCVLYRYGSFSV (SEQ ID NO: 388) amino acids 301-309 ofPRAME GVRIVVEYCLYVDSLFFL (SEQ ID NO: 389), LYVDSLFFLGVRIVVEYCLYVDSLFFL(SEQ ID NO: 390) amino acids 292-303 of MAGE-6 GVRIVVEYCKISGGPRISYPL(SEQ ID NO: 391), KISGGPRISYPLGVRIVVEYCKISGGPRISYPL (SEQ ID NO: 392)amino acids 157-167 of NY-ESO-1 GVRIVVEYCSLLMWITQCFL (SEQ ID NO: 393),SLLMWITQCFLGVRIVVEYCSLLMWITQCFL (SEQ ID NO: 394) amino acids 157-165 ofNY-ESO-1 GVRIVVEYCSLLMWITQC (SEQ ID NO: 395),SLLMWITQCGVRIVVEYCSLLMWITQC (SEQ ID NO: 396) amino acids 155-163 ofNY-ESO-1 GVRIVVEYCQLSLLMWIT (SEQ ID NO: 397),QLSLLMWITGVRIVVEYCQLSLLMWIT (SEQ ID NO: 398) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVF GVRIVVEYCTSYVKVLHHMVKISG (SEQ ID NO: 399)amino acids 281-295 of MAGE-3 I25 to C33 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 IVVEYCEPCEADPTGHSY (SEQ ID NO: 400), IVVEYCEPCEADPTGHSYIVVEYCEPCEADPTGHSY (SEQ ID NO: 401) amino acids 230-238 ofMAGE-1 IVVEYCEPCSAYGEPRKL (SEQ ID NO: 402), SAYGEPRKLIVVEYCEPCSAYGEPRKL(SEQ ID NO: 403) amino acids 168-176 of MAGE-3 IVVEYCEPCEVDPIGHLY (SEQID NO: 404), EVDPIGHLYIVVEYCEPCEVDPIGHLY (SEQ ID NO: 405) amino acids271-279 of MAGE-3 IVVEYCEPCFLWGPRALV (SEQ ID NO: 406),FLWGPRALVIVVEYCEPCFLWGPRALV (SEQ ID NO: 407) amino acids 167-176 ofMAGE-3 IVVEYCEPC MEVDPIGHLY (SEQ ID NO: 408),MEVDPIGHLYIVVEYCEPCMEVDPIGHLY (SEQ ID NO: 409) amino acids 2-10 of BAGEIVVEYCEPCAARAVFLAL (SEQ ID NO: 410), AARAVFLALIVVEYCEPCAARAVFLAL (SEQ IDNO: 411) amino acids 9-16 of GAGE-1,2 IVVEYCEPCYRPRPRRY (SEQ ID NO:412), YRPRPRRYIVVEYCEPCYRPRPRRY (SEQ ID NO: 413) amino acids 11-20 ofRAGE IVVEYCEPCSPSSNRIRNT (SEQ ID NO: 414), SPSSNRIRNTIVVEYCEPCSPSSNRIRNT(SEQ ID NO: 415) amino acids 23-32 of CDK4 IVVEYCEPCACDPHSGHFV (SEQ IDNO: 416), ACDPHSGHFVIVVEYCEPCACDPHSGHFV (SEQ ID NO: 417) amino acids29-37 of β-catenin IVVEYCEPCSYLDSGIHF (SEQ ID NO: 418),SYLDSGIHFIVVEYCEPCSYLDSGIHF (SEQ ID NO: 419) amino acids 1-9 oftyrosinase IVVEYCEPC MLLAVLYCL (SEQ ID NO: 420),MLLAVLYCLIVVEYCEPCMLLAVLYCL (SEQ ID NO: 421) amino acids 206-214 oftyrosinase IVVEYCEPC AFLPWHRLF (SEQ ID NO: 422),AFLPWHRLFIVVEYCEPCAFLPWHRLF (SEQ ID NO: 423) amino acids 56-70 oftyrosinase IVVEYCEPCQNILLSNAPLGPQFP (SEQ ID NO: 424),QNILLSNAPLGPQFPIVVEYCEPCQNILLSNAPLGPQFP (SEQ ID NO: 425) amino acids448-462 of tyrosinase IVVEYCEPCDYSYLQDSDPDSFQD (SEQ ID NO: 426),DYSYLQDSDPDSFQDIVVEYCEPCDYSYLQDSDPDSFQD (SEQ ID NO: 427) amino acids32-40 of Melan-A^(MART-1) IVVEYCEPCJLTVILGVL (SEQ ID NO: 428),JLTVILGVLIVVEYCEPCJLTVILGVL (SEQ ID NO: 429) amino acids 154-162 ofgp100^(Pme117) IVVEYCEPCKTWGQYWQV (SEQ ID NO: 430),KTWGQYWQVIVVEYCEPCKTWGQYWQV (SEQ ID NO: 431) amino acids 209-217 ofgp100^(Pme117) IVVEYCEPCITDQVPFSV (SEQ ID NO: 432),ITDQVPFSVIVVEYCEPCITDQVPFSV (SEQ ID NO: 433) amino acids 280-288 ofgp100^(Pme117) IVVEYCEPCYLEPGPVTA (SEQ ID NO: 434),YLEPGPVTAIVVEYCEPCYLEPGPVTA (SEQ ID NO: 435) amino acids 457-466 ofgp100^(Pme117) IVVEYCEPCLLDGTATLRL (SEQ ID NO: 436),LLDGTATLRLIVVEYCEPCLLDGTATLRL (SEQ ID NO: 437) amino acids 476-485 ofgp100^(Pme117) IVVEYCEPCVLYRYGSFSV (SEQ ID NO: 438),VLYRYGSFSVIVVEYCEPCVLYRYGSFSV (SEQ ID NO: 439) amino acids 301-309 ofPRAME IVVEYCEPCLYVDSLFFL (SEQ ID NO: 440), LYVDSLFFLIVVEYCEPCLYVDSLFFL(SEQ ID NO: 441) amino acids 292-303 of MAGE-6 IVVEYCEPCKISGGPRISYPL(SEQ ID NO: 442), KISGGPRISYPLIVVEYCEPCKISGGPRISYPL (SEQ ID NO: 443)amino acids 157-167 of NY-ESO-1 IVVEYCEPCSLLMWITQCFL (SEQ ID NO: 444),SLLMWITQCFLIVVEYCEPCSLLMWITQCFL (SEQ ID NO: 445) amino acids 157-165 ofNY-ESO-1 IVVEYCEPCSLLMWITQC (SEQ ID NO: 446),SLLMWITQCIVVEYCEPCSLLMWITQC (SEQ ID NO: 447) amino acids 155-163 ofNY-ESO-1 IVVEYCEPCQLSLLMWIT (SEQ ID NO: 448),QLSLLMWITIVVEYCEPCVQLSLLMWIT (SEQ ID NO: 449) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFIVVEYCEPCTSYVKVLHHMVKISG (SEQ ID NO: 450)amino acids 281-295 of MAGE-3 T38-V46 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 TYLELASAVEADPTGHSY (SEQ ID NO: 451), TYLELASAVEADPTGHSYTYLELASAVEADPTGHSY (SEQ ID NO: 452) amino acids 230-238 ofMAGE-1 TYLELASAVSAYGEPRKL (SEQ ID NO: 453), SAYGEPRKLTYLELASAVSAYGEPRKL(SEQ ID NO: 454) amino acids 168-176 of MAGE-3 TYLELASAVEVDPIGHLY (SEQID NO: 455), EVDPIGHLYTYLELASAVEVDPIGHLY (SEQ ID NO: 456) amino acids271-279 of MAGE-3 TYLELASAVFLWGPRALV (SEQ ID NO: 457),FLWGPRALVTYLELASAVFLWGPRALV (SEQ ID NO: 458) amino acids 167-176 ofMAGE-3 TYLELASAVMEVDPIGHLY (SEQ ID NO: 459),MEVDPIGHLYTYLELASAVMEVDPIGHLY (SEQ ID NO: 460) amino acids 2-10 of BAGETYLELASAVAARAVFLAL (SEQ ID NO: 461), AARAVFLALTYLELASAVAARAVFLAL (SEQ IDNO: 462) amino acids 9-16 of GAGE-1,2 TYLELASAVYRPRPRRY (SEQ ID NO:463), YRPRPRRYTYLELASAVYRPRPRRY (SEQ ID NO: 464) amino acids 11-20 ofRAGE TYLELASAVSPSSNRIRNT (SEQ ID NO: 465), SPSSNRIRNTTYLELASAVSPSSNRIRNT(SEQ ID NO: 466) amino acids 23-32 of CDK4 TYLELASAVACDPHSGHFV (SEQ IDNO: 467), ACDPHSGHFVTYLELASAVACDPHSGHFV (SEQ ID NO: 468) amino acids29-37 of β-catenin TYLELASAVSYLDSGIHF (SEQ ID NO: 469),SYLDSGIHFTYLELASAVSYLDSGIHF (SEQ ID NO: 470) amino acids 1-9 oftyrosinase TYLELASAVMLLAVLYCL (SEQ ID NO: 471),MLLAVLYCLTYLELASAVMLLAVLYCL (SEQ ID NO: 472) amino acids 206-214 oftyrosinase TYLELASAVAFLPWHRLF (SEQ ID NO: 473),AFLPWHRLFTYLELASAVAFLPWHRLF (SEQ ID NO: 474) amino acids 56-70 oftyrosinase TYLELASAVQNILLSNAPLGPQFP (SEQ ID NO: 475),QNILLSNAPLGPQFPTYLELASAVQNILLSNAPLGPQFP (SEQ ID NO: 476) amino acids448-462 of tyrosinase TYLELASAVDYSYLQDSDPDSFQD (SEQ ID NO: 477),DYSYLQDSDPDSFQDTYLELASAVDYSYLQDSDPDSFQD (SEQ ID NO: 478) amino acids32-40 of Melan-A^(MART-1) TYLELASAVJLTVILGVL (SEQ ID NO: 479),JLTVILGVLTYLELASAVJLTVILGVL (SEQ ID NO: 480) amino acids 154-162 ofgp100^(Pme117) TYLELASAVKTWGQYWQV (SEQ ID NO: 481),KTWGQYWQVTYLELASAVKTWGQYWQV (SEQ ID NO: 482) amino acids 209-217 ofgp100^(Pme117) TYLELASAVITDQVPFSV (SEQ ID NO: 483),ITDQVPFSVTYLELASAVITDQVPFSV (SEQ ID NO: 484) amino acids 280-288 ofgp100^(Pme117) TYLELASAVYLEPGPVTA (SEQ ID NO: 485),YLEPGPVTATYLELASAVYLEPGPVTA (SEQ ID NO: 486) amino acids 457-466 ofgp100^(Pme117) TYLELASAVLLDGTATLRL (SEQ ID NO: 487),LLDGTATLRLTYLELASAVLLDGTATLRL (SEQ ID NO: 488) amino acids 476-485 ofgp100^(Pme117) TYLELASAVVLYRYGSFSV (SEQ ID NO: 489),VLYRYGSFSVTYLELASAVVLYRYGSFSV (SEQ ID NO: 490) amino acids 301-309 ofPRAME TYLELASAVLYVDSLFFL (SEQ ID NO: 491), LYVDSLFFLTYLELASAVLYVDSLFFL(SEQ ID NO: 492) amino acids 292-303 of MAGE-6 TYLELASAVKISGGPRISYPL(SEQ ID NO: 493), KISGGPRISYPLTYLELASAVKISGGPRISYPL (SEQ ID NO: 494)amino acids 157-167 of NY-ESO-1 TYLELASAVSLLMWITQCFL (SEQ ID NO: 495),SLLMWITQCFLTYLELASAVSLLMWITQCFL (SEQ ID NO: 496) amino acids 157-165 ofNY-ESO-1 TYLELASAVSLLMWITQC (SEQ ID NO: 497),SLLMWITQCTYLELASAVSLLMWITQC (SEQ ID NO: 498) amino acids 155-163 ofNY-ESO-1 TYLELASAVQLSLLMWIT (SEQ ID NO: 499),QLSLLMWITTYLELASAVQLSLLMWIT (SEQ ID NO: 500) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVF TYLELASAVTSYVKVLHHMVKISG (SEQ ID NO: 501)amino acids 281-295 of MAGE-3 G61-I69 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 GTGAFEIEIEADPTGHSY (SEQ ID NO: 502), GTGAFEIEIEADPTGHSYGTGAFEIEIEADPTGHSY (SEQ ID NO: 503) amino acids 230-238 ofMAGE-1 GTGAFEIEISAYGEPRKL (SEQ ID NO: 504), SAYGEPRKLGTGAFEIEISAYGEPRKL(SEQ ID NO: 505) amino acids 168-176 of MAGE-3 GTGAFEIEIEVDPIGHLY (SEQID NO: 506), EVDPIGHLYGTGAFEIEIEVDPIGHLY (SEQ ID NO: 507) amino acids271-279 of MAGE-3 GTGAFEIEIFLWGPRALV (SEQ ID NO: 508),FLWGPRALVGTGAFEIEIFLWGPRALV (SEQ ID NO: 509) amino acids 167-176 ofMAGE-3 GTGAFEIEIMEVDPIGHLY (SEQ ID NO: 510),MEVDPIGHLYGTGAFEIEIMEVDPIGHLY (SEQ ID NO: 511) amino acids 2-10 of BAGEGTGAFEIEIAARAVFLAL (SEQ ID NO: 512), AARAVFLALGTGAFEIEIAARAVFLAL (SEQ IDNO: 513) amino acids 9-16 of GAGE-1,2 GTGAFEIEIYRPRPRRY (SEQ ID NO:514), YRPRPRRYGTGAFEIEIYRPRPRRY (SEQ ID NO: 515) amino acids 11-20 ofRAGE GTGAFEIEISPSSNRIRNT (SEQ ID NO: 516), SPSSNRIRNTGTGAFEIEISPSSNRIRNT(SEQ ID NO: 517) amino acids 23-32 of CDK4 GTGAFEIEIACDPHSGHFV (SEQ IDNO: 518), ACDPHSGHFVGTGAFEIEIACDPHSGHFV (SEQ ID NO: 519) amino acids29-37 of β-catenin GTGAFEIEISYLDSGIHF (SEQ ID NO: 520),SYLDSGIHFGTGAFEIEISYLDSGIHF (SEQ ID NO: 521) amino acids 1-9 oftyrosinase GTGAFEIEIMLLAVLYCL (SEQ ID NO: 522),MLLAVLYCLGTGAFEIEIMLLAVLYCL (SEQ ID NO: 523) amino acids 206-214 oftyrosinase GTGAFEIEIAFLPWHRLF (SEQ ID NO: 524),AFLPWHRLFGTGAFEIEIAFLPWHRLF (SEQ ID NO: 525) amino acids 56-70 oftyrosinase GTGAFEIEIQNILLSNAPLGPQFP (SEQ ID NO: 526),QNILLSNAPLGPQFPGTGAFEIEIQNILLSNAPLGPQFP (SEQ ID NO: 527) amino acids448-462 of tyrosinase GTGAFEIEIDYSYLQDSDPDSFQD (SEQ ID NO: 528),DYSYLQDSDPDSFQDGTGAFEIEIDYSYLQDSDPDSFQD (SEQ ID NO: 529) amino acids32-40 of Melan-A^(MART-1) GTGAFEIEIJLTVILGVL (SEQ ID NO: 530),JLTVILGVLGTGAFEIEIJLTVILGVL (SEQ ID NO: 531) amino acids 154-162 ofgp100^(Pme117) GTGAFEIEIKTWGQYWQV (SEQ ID NO: 532),KTWGQYWQVGTGAFEIEIKTWGQYWQV (SEQ ID NO: 533) amino acids 209-217 ofgp100^(Pme117) GTGAFEIEIITDQVPFSV (SEQ ID NO: 534),ITDQVPFSVGTGAFEIEIITDQVPFSV (SEQ ID NO: 535) amino acids 280-288 ofgp100^(Pme117) GTGAFEIEIYLEPGPVTA (SEQ ID NO: 536),YLEPGPVTAGTGAFEIEIYLEPGPVTA (SEQ ID NO: 537) amino acids 457-466 ofgp100^(Pme117) GTGAFEIEILLDGTATLRL (SEQ ID NO: 538),LLDGTATLRLGTGAFEIEILLDGTATLRL (SEQ ID NO: 539) amino acids 476-485 ofgp100^(Pme117) GTGAFEIEIVLYRYGSFSV (SEQ ID NO: 540),VLYRYGSFSVGTGAFEIEIVLYRYGSFSV (SEQ ID NO: 541) amino acids 301-309 ofPRAME GTGAFEIEILYVDSLFFL (SEQ ID NO: 542), LYVDSLFFLGTGAFEIEILYVDSLFFL(SEQ ID NO: 543) amino acids 292-303 of MAGE-6 GTGAFEIEIKISGGPRISYPL(SEQ ID NO: 544), KISGGPRISYPLGTGAFEIEIKISGGPRISYPL (SEQ ID NO: 545)amino acids 157-167 of NY-ESO-1 GTGAFEIEISLLMWITQCFL (SEQ ID NO: 546),SLLMWITQCFLGTGAFEIEISLLMWITQCFL (SEQ ID NO: 547) amino acids 157-165 ofNY-ESO-1 GTGAFEIEISLLMWITQC (SEQ ID NO: 548),SLLMWITQCGTGAFEIEISLLMWITQC (SEQ ID NO: 549) amino acids 155-163 ofNY-ESO-1 GTGAFEIEIQLSLLMWIT (SEQ ID NO: 550),QLSLLMWITGTGAFEIEIQLSLLMWIT (SEQ ID NO: 551) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFGTGAFEIEITSYVKVLHHMVKISG (SEQ ID NO: 552)amino acids 281-295 of MAGE-3 F65-L73 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 FEIEINGQLEADPTGHSY (SEQ ID NO: 553), FEIEINGQLEADPTGHSYFEIEINGQLEADPTGHSY (SEQ ID NO: 554) amino acids 230-238 ofMAGE-1 FEIEINGQLSAYGEPRKL (SEQ ID NO: 555), SAYGEPRKLFEIEINGQLSAYGEPRKL(SEQ ID NO: 556) amino acids 168-176 of MAGE-3 FEIEINGQLEVDPIGHLY (SEQID NO: 557), EVDPIGHLYFEIEINGQLEVDPIGHLY (SEQ ID NO: 558) amino acids271-279 of MAGE-3 FEIEINGQLFLWGPRALV (SEQ ID NO: 559),FLWGPRALVFEIEINGQLFLWGPRALV (SEQ ID NO: 560) amino acids 167-176 ofMAGE-3 FEIEINGQLMEVDPIGHLY (SEQ ID NO: 561),MEVDPIGHLYFEIEINGQLMEVDPIGHLY (SEQ ID NO: 562) amino acids 2-10 of BAGEFEIEINGQLAARAVFLAL (SEQ ID NO: 563), AARAVFLALFEIEINGQLAARAVFLAL (SEQ IDNO: 564) amino acids 9-16 of GAGE-1,2 FEIEINGQLYRPRPRRY (SEQ ID NO:565), YRPRPRRYFEIEINGQLYRPRPRRY (SEQ ID NO: 566) amino acids 11-20 ofRAGE FEIEINGQLSPSSNRIRNT (SEQ ID NO: 567), SPSSNRIRNTFEIEINGQLSPSSNRIRNT(SEQ ID NO: 568) amino acids 23-32 of CDK4 FEIEINGQLACDPHSGHFV (SEQ IDNO: 569), ACDPHSGHFVFEIEINGQLACDPHSGHFV (SEQ ID NO: 570) amino acids29-37 of β-catenin FEIEINGQLSYLDSGIHF (SEQ ID NO: 571),SYLDSGIHFFEIEINGQLSYLDSGIHF (SEQ ID NO: 572) amino acids 1-9 oftyrosinase FEIEINGQLMLLAVLYCL (SEQ ID NO: 573),MLLAVLYCLFEIEINGQLMLLAVLYCL (SEQ ID NO: 574) amino acids 206-214 oftyrosinase FEIEINGQL AFLPWHRLF (SEQ ID NO: 575),AFLPWHRLFFEIEINGQLAFLPWHRLF (SEQ ID NO: 576) amino acids 56-70 oftyrosinase FEIEINGQLQNILLSNAPLGPQFP (SEQ ID NO: 577),QNILLSNAPLGPQFPFEIEINGQLQNILLSNAPLGPQFP (SEQ ID NO: 578) amino acids448-462 of tyrosinase FEIEINGQLDYSYLQDSDPDSFQD (SEQ ID NO: 579),DYSYLQDSDPDSFQDFEIEINGQLDYSYLQDSDPDSFQD (SEQ ID NO: 580) amino acids32-40 of Melan-A^(MART-1) FEIEINGQLJLTVILGVL (SEQ ID NO: 581),JLTVILGVLFEIEINGQLJLTVILGVL (SEQ ID NO: 582) amino acids 154-162 ofgp100^(Pme117) FEIEINGQLKTWGQYWQV (SEQ ID NO: 583),KTWGQYWQVFEIEINGQLKTWGQYWQV (SEQ ID NO: 584) amino acids 209-217 ofgp100^(Pme117) FEIEINGQLITDQVPFSV (SEQ ID NO: 585),ITDQVPFSVFEIEINGQLITDQVPFSV (SEQ ID NO: 586) amino acids 280-288 ofgp100^(Pme117) FEIEINGQLYLEPGPVTA (SEQ ID NO: 587),YLEPGPVTAFEIEINGQLYLEPGPVTA (SEQ ID NO: 588) amino acids 457-466 ofgp100^(Pme117) FEIEINGQLLLDGTATLRL (SEQ ID NO: 589),LLDGTATLRLFEIEINGQLLLDGTATLRL (SEQ ID NO: 590) amino acids 476-485 ofgp100^(Pme117) FEIEINGQLVLYRYGSFSV (SEQ ID NO: 591),VLYRYGSFSVFEIEINGQLVLYRYGSFSV (SEQ ID NO: 592) amino acids 301-309 ofPRAME FEIEINGQLLYVDSLFFL (SEQ ID NO: 593), LYVDSLFFLFEIEINGQLLYVDSLFFL(SEQ ID NO: 594) amino acids 292-303 of MAGE-6 FEIEINGQLKISGGPRISYPL(SEQ ID NO: 595), KISGGPRISYPLFEIEINGQLKISGGPRISYPL (SEQ ID NO: 596)amino acids 157-167 of NY-ESO-1 FEIEINGQLSLLMWITQCFL (SEQ ID NO: 597),SLLMWITQCFLFEIEINGQLSLLMWITQCFL (SEQ ID NO: 598) amino acids 157-165 ofNY-ESO-1 FEIEINGQLSLLMWITQC (SEQ ID NO: 599),SLLMWITQCFEIEINGQLSLLMWITQC (SEQ ID NO: 600) amino acids 155-163 ofNY-ESO-1 FEIEINGQLQLSLLMWIT (SEQ ID NO: 601),QLSLLMWITFEIEINGQLQLSLLMWIT (SEQ ID NO: 602) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFFEIEINGQLTSYVKVLHHMVKISG (SEQ ID NO: 603)amino acids 281-295 of MAGE-3 I67-F75 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 IEINGQLVFEADPTGHSY (SEQ ID NO: 604), IEINGQLVFEADPTGHSYIEINGQLVFEADPTGHSY (SEQ ID NO: 605) amino acids 230-238 ofMAGE-1 IEINGQLVFSAYGEPRKL (SEQ ID NO: 606), SAYGEPRKLIEINGQLVFSAYGEPRKL(SEQ ID NO: 607) amino acids 168-176 of MAGE-3 IEINGQLVFEVDPIGHLY (SEQID NO: 608), EVDPIGHLYIEINGQLVFEVDPIGHLY (SEQ ID NO: 609) amino acids271-279 of MAGE-3 IEINGQLVFFLWGPRALV (SEQ ID NO: 610),FLWGPRALVIEINGQLVFFLWGPRALV (SEQ ID NO: 611) amino acids 167-176 ofMAGE-3 IEINGQLVFMEVDPIGHLY (SEQ ID NO: 612),MEVDPIGHLYIEINGQLVFMEVDPIGHLY (SEQ ID NO: 613) amino acids 2-10 of BAGEIEINGQLVFAARAVFLAL (SEQ ID NO: 614), AARAVFLALIEINGQLVFAARAVFLAL (SEQ IDNO: 615) amino acids 9-16 of GAGE-1,2 IEINGQLVFYRPRPRRY (SEQ ID NO:616), YRPRPRRYIEINGQLVFYRPRPRRY (SEQ ID NO: 617) amino acids 11-20 ofRAGE IEINGQLVFSPSSNRIRNT (SEQ ID NO: 618), SPSSNRIRNTIEINGQLVFSPSSNRIRNT(SEQ ID NO: 619) amino acids 23-32 of CDK4 IEINGQLVFCDPHSGHFV (SEQ IDNO: 620), ACDPHSGHFVIEINGQLVFACDPHSGHFV (SEQ ID NO: 621) amino acids29-37 of β-catenin IEINGQLVFSYLDSGIHF (SEQ ID NO: 622),SYLDSGIHFIEINGQLVFSYLDSGIHF (SEQ ID NO: 623) amino acids 1-9 oftyrosinase IEINGQLVFMLLAVLYCL (SEQ ID NO: 624),MLLAVLYCLIEINGQLVFMLLAVLYCL (SEQ ID NO: 625) amino acids 206-214 oftyrosinase IEINGQLVF AFLPWHRLF (SEQ ID NO: 626),AFLPWHRLFIEINGQLVFAFLPWHRLF (SEQ ID NO: 627) amino acids 56-70 oftyrosinase IEINGQLVFQNILLSNAPLGPQFP (SEQ ID NO: 628),QNILLSNAPLGPQFPIEINGQLVFQNILLSNAPLGPQFP (SEQ ID NO: 629) amino acids448-462 of tyrosinase IEINGQLVFDYSYLQDSDPDSFQD (SEQ ID NO: 630),DYSYLQDSDPDSFQDIEINGQLVFDYSYLQDSDPDSFQD (SEQ ID NO: 631) amino acids32-40 of Melan-A^(MART-1) IEINGQLVFJLTVILGVL (SEQ ID NO: 632),JLTVILGVLIEINGQLVFJLTVILGVL (SEQ ID NO: 633) amino acids 154-162 ofgp100^(Pme117) IEINGQLVFKTWGQYWQV (SEQ ID NO: 634),KTWGQYWQVIEINGQLVFKTWGQYWQV (SEQ ID NO: 635) amino acids 209-217 ofgp100^(Pme117) IEINGQLVFITDQVPFSV (SEQ ID NO: 636),ITDQVPFSVIEINGQLVFITDQVPFSV (SEQ ID NO: 637) amino acids 280-288 ofgp100^(Pme117) IEINGQLVFYLEPGPVTA (SEQ ID NO: 638),YLEPGPVTAIEINGQLVFYLEPGPVTA (SEQ ID NO: 639) amino acids 457-466 ofgp100^(Pme117) IEINGQLVFLLDGTATLRL (SEQ ID NO: 640),LLDGTATLRLIEINGQLVFLLDGTATLRL (SEQ ID NO: 641) amino acids 476-485 ofgp100^(Pme117) IEINGQLVFVLYRYGSFSV (SEQ ID NO: 642),VLYRYGSFSVIEINGQLVFVLYRYGSFSV (SEQ ID NO: 643) amino acids 301-309 ofPRAME IEINGQLVFLYVDSLFFL (SEQ ID NO: 644), LYVDSLFFLIEINGQLVFLYVDSLFFL(SEQ ID NO: 645) amino acids 292-303 of MAGE-6 IEINGQLVFKISGGPRISYPL(SEQ ID NO: 646), KISGGPRISYPLIEINGQLVFKISGGPRISYPL (SEQ ID NO: 647)amino acids 157-167 of NY-ESO-1 IEINGQLVFSLLMWITQCFL (SEQ ID NO: 648),SLLMWITQCFLIEINGQLVFSLLMWITQCFL (SEQ ID NO: 649) amino acids 157-165 ofNY-ESO-1 IEINGQLVFSLLMWITQC (SEQ ID NO: 650),SLLMWITQCIEINGQLVFSLLMWITQC (SEQ ID NO: 651) amino acids 155-163 ofNY-ESO-1 IEINGQLVFQLSLLMWIT (SEQ ID NO: 652),QLSLLMWITIEINGQLVFQLSLLMWIT (SEQ ID NO: 653) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFIEINGQLVFTSYVKVLHHMVKISG (SEQ ID NO: 654)amino acids 281-295 of MAGE-3 K77-Y85 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 KLENGGFPYEADPTGHSY (SEQ ID NO: 655), KLENGGFPYEADPTGHSYKLENGGFPYEADPTGHSY (SEQ ID NO: 656) amino acids 230-238 ofMAGE-1 KLENGGFPYSAYGEPRKL (SEQ ID NO: 657), SAYGEPRKLKLENGGFPYSAYGEPRKL(SEQ ID NO: 658) amino acids 168-176 of MAGE-3 KLENGGFPYEVDPIGHLY (SEQID NO: 659), EVDPIGHLYKLENGGFPYEVDPIGHLY (SEQ ID NO: 660) amino acids271-279 of MAGE-3 KLENGGFPYFLWGPRALV (SEQ ID NO: 661),FLWGPRALVKLENGGFPYFLWGPRALV (SEQ ID NO: 662) amino acids 167-176 ofMAGE-3 KLENGGFPYMEVDPIGHLY (SEQ ID NO: 663),MEVDPIGHLYKLENGGFPYMEVDPIGHLY (SEQ ID NO: 664) amino acids 2-10 of BAGEKLENGGFPYAARAVFLAL (SEQ ID NO: 665), AARAVFLALKLENGGFPYAARAVFLAL (SEQ IDNO: 666) amino acids 9-16 of GAGE-1,2 KLENGGFPYYRPRPRRY (SEQ ID NO:667), YRPRPRRYKLENGGFPYYRPRPRRY (SEQ ID NO: 668) amino acids 11-20 ofRAGE KLENGGFPYSPSSNRIRNT (SEQ ID NO: 669), SPSSNRIRNTKLENGGFPYSPSSNRIRNT(SEQ ID NO: 670) amino acids 23-32 of CDK4 KLENGGFPYACDPHSGHFV (SEQ IDNO: 671), ACDPHSGHFVKLENGGFPYACDPHSGHFV (SEQ ID NO: 672) amino acids29-37 of β-catenin KLENGGFPYSYLDSGIHF (SEQ ID NO: 673),SYLDSGIHFKLENGGFPYSYLDSGIHF (SEQ ID NO: 674) amino acids 1-9 oftyrosinase KLENGGFPYMLLAVLYCL (SEQ ID NO: 675),MLLAVLYCLKLENGGFPYMLLAVLYCL (SEQ ID NO: 676) amino acids 206-214 oftyrosinase KLENGGFPYAFLPWHRLF (SEQ ID NO: 677),AFLPWHRLFKLENGGFPYAFLPWHRLF (SEQ ID NO: 678) amino acids 56-70 oftyrosinase KLENGGFPYQNILLSNAPLGPQFP (SEQ ID NO: 679),QNILLSNAPLGPQFPKLENGGFPYQNILLSNAPLGPQFP (SEQ ID NO: 680) amino acids448-462 of tyrosinase KLENGGFPYDYSYLQDSDPDSFQD (SEQ ID NO: 681),DYSYLQDSDPDSFQDKLENGGFPYDYSYLQDSDPDSFQD (SEQ ID NO: 682) amino acids32-40 of Melan-A^(MART-1) KLENGGFPYJLTVILGVL (SEQ ID NO: 683),JLTVILGVLKLENGGFPYJLTVILGVL (SEQ ID NO: 684) amino acids 154-162 ofgp100^(Pme117) KLENGGFPYKTWGQYWQV (SEQ ID NO: 685),KTWGQYWQVKLENGGFPYKTWGQYWQV (SEQ ID NO: 686) amino acids 209-217 ofgp100^(Pme117) KLENGGFPYITDQVPFSV (SEQ ID NO: 687),ITDQVPFSVKLENGGFPYITDQVPFSV (SEQ ID NO: 688) amino acids 280-288 ofgp100^(Pme117) KLENGGFPYYLEPGPVTA (SEQ ID NO: 689),YLEPGPVTAKLENGGFPYYLEPGPVTA (SEQ ID NO: 690) amino acids 457-466 ofgp100^(Pme117) KLENGGFPYLLDGTATLRL (SEQ ID NO: 691),LLDGTATLRLKLENGGFPYLLDGTATLRL (SEQ ID NO: 692) amino acids 476-485 ofgp100^(Pme117) KLENGGFPYVLYRYGSFSV (SEQ ID NO: 693),VLYRYGSFSVKLENGGFPYVLYRYGSFSV (SEQ ID NO: 694) amino acids 301-309 ofPRAME KLENGGFPYLYVDSLFFL (SEQ ID NO: 695), LYVDSLFFLKLENGGFPYLYVDSLFFL(SEQ ID NO: 696) amino acids 292-303 of MAGE-6 KLENGGFPYKISGGPRISYPL(SEQ ID NO: 697), KISGGPRISYPLKLENGGFPYKISGGPRISYPL (SEQ ID NO: 698)amino acids 157-167 of NY-ESO-1 KLENGGFPYSLLMWITQCFL (SEQ ID NO: 699),SLLMWITQCFLKLENGGFPYSLLMWITQCFL (SEQ ID NO: 700) amino acids 157-165 ofNY-ESO-1 KLENGGFPYSLLMWITQC (SEQ ID NO: 701),SLLMWITQCKLENGGFPYSLLMWITQC (SEQ ID NO: 702) amino acids 155-163 ofNY-ESO-1 KLENGGFPYQLSLLMWIT (SEQ ID NO: 703),QLSLLMWITKLENGGFPYQLSLLMWIT (SEQ ID NO: 704) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVF KLENGGFPYTSYVKVLHHMVKISG (SEQ ID NO: 705)amino acids 281-295 of MAGE-3 Q72-E86 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 QLVFSKLENGGFPYEEADPTGHSY (SEQ ID NO: 706),QLVFSKLENGGFPYE EADPTGHSYQLVFSKLENGGFPYEEADPTGHSY (SEQ ID NO: 707) aminoacids 230-238 of MAGE-1 QLVFSKLENGGFPYESAYGEPRKL (SEQ ID NO: 708),SAYGEPRKLQLVFSKLENGGFPYESAYGEPRKL (SEQ ID NO: 709) amino acids 168-176of MAGE-3 QLVFSKLENGGFPYEEVDPIGHLY (SEQ ID NO: 710),EVDPIGHLYQLVFSKLENGGFPYEEVDPIGHLY (SEQ ID NO: 711) amino acids 271-279of MAGE-3 QLVFSKLENGGFPYEFLWGPRALV (SEQ ID NO: 712),FLWGPRALVQLVFSKLENGGFPYEFLWGPRALV (SEQ ID NO: 713) amino acids 167-176of MAGE-3 QLVFSKLENGGFPYEMEVDPIGHLY (SEQ ID NO: 714),MEVDPIGHLYQLVFSKLENGGFPYEMEVDPIGHLY (SEQ ID NO: 715) amino acids 2-10 ofBAGE QLVFSKLENGGFPYEAARAVFLAL (SEQ ID NO: 716),AARAVFLALQLVFSKLENGGFPYEAARAVFLAL (SEQ ID NO: 717) amino acids 9-16 ofGAGE-1,2 QLVFSKLENGGFPYEYRPRPRRY (SEQ ID NO: 718),YRPRPRRYQLVFSKLENGGFPYEYRPRPRRY (SEQ ID NO: 719) amino acids 11-20 ofRAGE QLVFSKLENGGFPYESPSSNRIRNT (SEQ ID NO: 720),SPSSNRIRNTQLVFSKLENGGFPYESPSSNRIRNT (SEQ ID NO: 721) amino acids 23-32of CDK4 QLVFSKLENGGFPYEACDPHSGHFV (SEQ ID NO: 722),ACDPHSGHFVQLVFSKLENGGFPYEACDPHSGHFV (SEQ ID NO: 723) amino acids 29-37of β-catenin QLVFSKLENGGFPYESYLDSGIHF (SEQ ID NO: 724),SYLDSGIHFQLVFSKLENGGFPYESYLDSGIHF (SEQ ID NO: 725) amino acids 1-9 oftyrosinase QLVFSKLENGGFPYEMLLAVLYCL (SEQ ID NO: 726),MLLAVLYCLQLVFSKLENGGFPYEMLLAVLYCL (SEQ ID NO: 727) amino acids 206-214of tyrosinase QLVFSKLENGGFPYEAFLPWHRLF (SEQ ID NO: 728),AFLPWHRLFQLVFSKLENGGFPYEAFLPWHRLF (SEQ ID NO: 729) amino acids 56-70 oftyrosinase QLVFSKLENGGFPYEQNILLSNAPLGPQFP (SEQ ID NO: 730),QNILLSNAPLGPQFPQLVFSKLENGGFPYEQNILLSNAPLGPQFP (SEQ ID NO: 731) aminoacids 448-462 of tyrosinase QLVFSKLENGGFPYEDYSYLQDSDPDSFQD (SEQ ID NO:732), DYSYLQDSDPDSFQDQLVFSKLENGGFPYEDYSYLQDSDPDSFQD (SEQ ID NO: 733)amino acids 32-40 of Melan-A^(MART-1) QLVFSKLENGGFPYEJLTVILGVL (SEQ IDNO: 734), JLTVILGVLQLVFSKLENGGFPYEJLTVILGVL (SEQ ID NO: 735) amino acids154-162 of gp100^(Pme117) QLVFSKLENGGFPYEKTWGQYWQV (SEQ ID NO: 736),KTWGQYWQVQLVFSKLENGGFPYEKTWGQYWQV (SEQ ID NO: 737) amino acids 209-217of gp100^(Pme117) QLVFSKLENGGFPYEITDQVPFSV (SEQ ID NO: 738),ITDQVPFSVQLVFSKLENGGFPYEITDQVPFSV (SEQ ID NO: 739) amino acids 280-288of gp100^(Pme117) QLVFSKLENGGFPYEYLEPGPVTA (SEQ ID NO: 740),YLEPGPVTAQLVFSKLENGGFPYEYLEPGPVTA (SEQ ID NO: 741) amino acids 457-466of gp100^(Pme117) QLVFSKLENGGFPYELLDGTATLRL (SEQ ID NO: 742),LLDGTATLRLQLVFSKLENGGFPYELLDGTATLRL (SEQ ID NO: 743) amino acids 476-485of gp100^(Pme117) QLVFSKLENGGFPYEVLYRYGSFSV (SEQ ID NO: 744),VLYRYGSFSVQLVFSKLENGGFPYEVLYRYGSFSV (SEQ ID NO: 745) amino acids 301-309of PRAME QLVFSKLENGGFPYELYVDSLFFL (SEQ ID NO: 746),LYVDSLFFLQLVFSKLENGGFPYELYVDSLFFL (SEQ ID NO: 747) amino acids 292-303of MAGE-6 QLVFSKLENGGFPYEKISGGPRISYPL (SEQ ID NO: 748),KISGGPRISYPLQLVFSKLENGGFPYEKISGGPRISYPL (SEQ ID NO: 749) amino acids157-167 of NY-ESO-1 QLVFSKLENGGFPYESLLMWITQCFL (SEQ ID NO: 750),SLLMWITQCFLQLVFSKLENGGFPYESLLMWITQCFL (SEQ ID NO: 751) amino acids157-165 of NY-ESO-1 QLVFSKLENGGFPYESLLMWITQC (SEQ ID NO: 752),SLLMWITQCQLVFSKLENGGFPYESLLMWITQC (SEQ ID NO: 753) amino acids 155-163of NY-ESO-1 QLVFSKLENGGFPYEQLSLLMWIT (SEQ ID NO: 754),QLSLLMWITQLVFSKLENGGFPYEQLSLLMWIT (SEQ ID NO: 755) amino acids 157-170of NY-ESO-1 and SLLMWITQCFLPVFQLVFSKLENGGFPYETSYVKVLHHMVKISG (SEQ ID NO:756) amino acids 281-295 of MAGE-3 G81-L89 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 GGFPYEKDLEADPTGHSY (SEQ ID NO: 757), GGFPYEKDLEADPTGHSYGGFPYEKDLEADPTGHSY (SEQ ID NO: 758) amino acids 230-238 ofMAGE-1 GGFPYEKDLSAYGEPRKL (SEQ ID NO: 759), SAYGEPRKLGGFPYEKDLSAYGEPRKL(SEQ ID NO: 760) amino acids 168-176 of MAGE-3 GGFPYEKDLEVDPIGHLY (SEQID NO: 761), EVDPIGHLYGGFPYEKDLEVDPIGHLY (SEQ ID NO: 762) amino acids271-279 of MAGE-3 GGFPYEKDLFLWGPRALV (SEQ ID NO: 763),FLWGPRALVGGFPYEKDLFLWGPRALV (SEQ ID NO: 764) amino acids 167-176 ofMAGE-3 GGFPYEKDLMEVDPIGHLY (SEQ ID NO: 765),MEVDPIGHLYGGFPYEKDLMEVDPIGHLY (SEQ ID NO: 766) amino acids 2-10 of BAGEGGFPYEKDLAARAVFLAL (SEQ ID NO: 767), AARAVFLALGGFPYEKDLAARAVFLAL (SEQ IDNO: 768) amino acids 9-16 of GAGE-1,2 GGFPYEKDLYRPRPRRY (SEQ ID NO:769), YRPRPRRYGGFPYEKDLYRPRPRRY (SEQ ID NO: 770) amino acids 11-20 ofRAGE GGFPYEKDLSPSSNRIRNT (SEQ ID NO: 771), SPSSNRIRNTGGFPYEKDLSPSSNRIRNT(SEQ ID NO: 772) amino acids 23-32 of CDK4 GGFPYEKDLACDPHSGHFV (SEQ IDNO: 773), ACDPHSGHFVGGFPYEKDLACDPHSGHFV (SEQ ID NO: 774) amino acids29-37 of β-catenin GGFPYEKDLSYLDSGIHF (SEQ ID NO: 775),SYLDSGIHFGGFPYEKDLSYLDSGIHF (SEQ ID NO: 776) amino acids 1-9 oftyrosinase GGFPYEKDLMLLAVLYCL (SEQ ID NO: 777),MLLAVLYCLGGFPYEKDLMLLAVLYCL (SEQ ID NO: 778) amino acids 206-214 oftyrosinase GGFPYEKDL AFLPWHRLF (SEQ ID NO: 779),AFLPWHRLFGGFPYEKDLAFLPWHRLF (SEQ ID NO: 780) amino acids 56-70 oftyrosinase GGFPYEKDLQNILLSNAPLGPQFP (SEQ ID NO: 781),QNILLSNAPLGPQFPGGFPYEKDLQNILLSNAPLGPQFP (SEQ ID NO: 782) amino acids448-462 of tyrosinase GGFPYEKDLDYSYLQDSDPDSFQD (SEQ ID NO: 783),DYSYLQDSDPDSFQDGGFPYEKDLDYSYLQDSDPDSFQD (SEQ ID NO: 784) amino acids32-40 of Melan-A^(MART-1) GGFPYEKDLJLTVILGVL (SEQ ID NO: 785),JLTVILGVLGGFPYEKDLJLTVILGVL (SEQ ID NO: 786) amino acids 154-162 ofgp100^(Pme117) GGFPYEKDLKTWGQYWQV (SEQ ID NO: 787),KTWGQYWQVGGFPYEKDLKTWGQYWQV (SEQ ID NO: 788) amino acids 209-217 ofgp100^(Pme117) GGFPYEKDLITDQVPFSV (SEQ ID NO: 789),ITDQVPFSVGGFPYEKDLITDQVPFSV (SEQ ID NO: 790) amino acids 280-288 ofgp100^(Pme117) GGFPYEKDLYLEPGPVTA (SEQ ID NO: 791),YLEPGPVTAGGFPYEKDLYLEPGPVTA (SEQ ID NO: 792) amino acids 457-466 ofgp100^(Pme117) GGFPYEKDLLLDGTATLRL (SEQ ID NO: 793),LLDGTATLRLGGFPYEKDLLLDGTATLRL (SEQ ID NO: 794) amino acids 476-485 ofgp100^(Pme117) GGFPYEKDLVLYRYGSFSV (SEQ ID NO: 795),VLYRYGSFSVGGFPYEKDLVLYRYGSFSV (SEQ ID NO: 796) amino acids 301-309 ofPRAME GGFPYEKDLLYVDSLFFL (SEQ ID NO: 797), LYVDSLFFLGGFPYEKDLLYVDSLFFL(SEQ ID NO: 798) amino acids 292-303 of MAGE-6 GGFPYEKDLKISGGPRISYPL(SEQ ID NO: 799), KISGGPRISYPLGGFPYEKDLKISGGPRISYPL (SEQ ID NO: 800)amino acids 157-167 of NY-ESO-1 GGFPYEKDLSLLMWITQCFL (SEQ ID NO: 801),SLLMWITQCFLGGFPYEKDLSLLMWITQCFL (SEQ ID NO: 802) amino acids 157-165 ofNY-ESO-1 GGFPYEKDLSLLMWITQC (SEQ ID NO: 803),SLLMWITQCGGFPYEKDLSLLMWITQC (SEQ ID NO: 804) amino acids 155-163 ofNY-ESO-1 GGFPYEKDLQLSLLMWIT (SEQ ID NO: 805),QLSLLMWITGGFPYEKDLQLSLLMWIT (SEQ ID NO: 806) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFGGFPYEKDLTSYVKVLHHMVKISG (SEQ ID NO: 807)amino acids 281-295 of MAGE-3 K104-C112 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 KITNSRPPCEADPTGHSY (SEQ ID NO: 808), KITNSRPPCEADPTGHSYKITNSRPPCEADPTGHSY (SEQ ID NO: 809) amino acids 230-238 ofMAGE-1 KITNSRPPCSAYGEPRKL (SEQ ID NO: 810), SAYGEPRKLKITNSRPPCSAYGEPRKL(SEQ ID NO: 811) amino acids 168-176 of MAGE-3 KITNSRPPCEVDPIGHLY (SEQID NO: 812), EVDPIGHLYKITNSRPPCEVDPIGHLY (SEQ ID NO: 813) amino acids271-279 of MAGE-3 KITNSRPPCFLWGPRALV (SEQ ID NO: 814),FLWGPRALVKITNSRPPCFLWGPRALV (SEQ ID NO: 815) amino acids 167-176 ofMAGE-3 KITNSRPPCMEVDPIGHLY (SEQ ID NO: 816),MEVDPIGHLYKITNSRPPCMEVDPIGHLY (SEQ ID NO: 817) amino acids 2-10 of BAGEKITNSRPPCAARAVFLAL (SEQ ID NO: 818), AARAVFLALKITNSRPPCAARAVFLAL (SEQ IDNO: 819) amino acids 9-16 of GAGE-1,2 KITNSRPPCYRPRPRRY (SEQ ID NO:820), YRPRPRRYKITNSRPPCYRPRPRRY (SEQ ID NO: 821) amino acids 11-20 ofRAGE KITNSRPPCSPSSNRIRNT (SEQ ID NO: 822), SPSSNRIRNTKITNSRPPCSPSSNRIRNT(SEQ ID NO: 823) amino acids 23-32 of CDK4 KITNSRPPCACDPHSGHFV (SEQ IDNO: 824), ACDPHSGHFVKITNSRPPCACDPHSGHFV (SEQ ID NO: 825) amino acids29-37 of β-catenin KITNSRPPCSYLDSGIHF (SEQ ID NO: 826),SYLDSGIHFKITNSRPPCSYLDSGIHF (SEQ ID NO: 827) amino acids 1-9 oftyrosinase KITNSRPPCMLLAVLYCL (SEQ ID NO: 828),MLLAVLYCLKITNSRPPCMLLAVLYCL (SEQ ID NO: 829) amino acids 206-214 oftyrosinase KITNSRPPCAFLPWHRLF (SEQ ID NO: 830),AFLPWHRLFKITNSRPPCAFLPWHRLF (SEQ ID NO: 831) amino acids 56-70 oftyrosinase KITNSRPPCQNILLSNAPLGPQFP (SEQ ID NO: 832),QNILLSNAPLGPQFPKITNSRPPCQNILLSNAPLGPQFP (SEQ ID NO: 833) amino acids448-462 of tyrosinase KITNSRPPCDYSYLQDSDPDSFQD (SEQ ID NO: 834),DYSYLQDSDPDSFQDKITNSRPPCDYSYLQDSDPDSFQD (SEQ ID NO: 835) amino acids32-40 of Melan-A^(MART-1) KITNSRPPCJLTVILGVL (SEQ ID NO: 836),JLTVILGVLKITNSRPPCJLTVILGVL (SEQ ID NO: 837) amino acids 154-162 ofgp100^(Pme117) KITNSRPPCKTWGQYWQV (SEQ ID NO: 838),KTWGQYWQVKITNSRPPCKTWGQYWQV (SEQ ID NO: 839) amino acids 209-217 ofgp100^(Pme117) KITNSRPPCITDQVPFSV (SEQ ID NO: 840),ITDQVPFSVKITNSRPPCITDQVPFSV (SEQ ID NO: 841) amino acids 280-288 ofgp100^(Pme117) KITNSRPPCYLEPGPVTA (SEQ ID NO: 842),YLEPGPVTAKITNSRPPCYLEPGPVTA (SEQ ID NO: 843) amino acids 457-466 ofgp100^(Pme117) KITNSRPPCLLDGTATLRL (SEQ ID NO: 844),LLDGTATLRLKITNSRPPCLLDGTATLRL (SEQ ID NO: 845) amino acids 476-485 ofgp100^(Pme117) KITNSRPPCVLYRYGSFSV (SEQ ID NO: 846),VLYRYGSFSVKITNSRPPCVLYRYGSFSV (SEQ ID NO: 847) amino acids 301-309 ofPRAME KITNSRPPCLYVDSLFFL (SEQ ID NO: 848), LYVDSLFFLKITNSRPPCLYVDSLFFL(SEQ ID NO: 849) amino acids 292-303 of MAGE-6 KITNSRPPCKISGGPRISYPL(SEQ ID NO: 850), KISGGPRISYPLKITNSRPPCKISGGPRISYPL (SEQ ID NO: 851)amino acids 157-167 of NY-ESO-1 KITNSRPPCSLLMWITQCFL (SEQ ID NO: 852),SLLMWITQCFLKITNSRPPCSLLMWITQCFL (SEQ ID NO: 853) amino acids 157-165 ofNY-ESO-1 KITNSRPPCSLLMWITQC (SEQ ID NO: 854),SLLMWITQCKITNSRPPCSLLMWITQC (SEQ ID NO: 855) amino acids 155-163 ofNY-ESO-1 KITNSRPPCQLSLLMWIT (SEQ ID NO: 856),QLSLLMWITKITNSRPPCQLSLLMWIT (SEQ ID NO: 857) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFKITNSRPPCTSYVKVLHHMVKISG (SEQ ID NO: 858)amino acids 281-295 of MAGE-3 K104-V113 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 KITNSRPPCVEADPTGHSY (SEQ ID NO: 859), KITNSRPPCVEADPTGHSYKITNSRPPCVEADPTGHSY (SEQ ID NO: 860) amino acids 230-238 ofMAGE-1 KITNSRPPCVSAYGEPRKL (SEQ ID NO: 861),SAYGEPRKLKITNSRPPCVSAYGEPRKL (SEQ ID NO: 862) amino acids 168-176 ofMAGE-3 KITNSRPPCVEVDPIGHLY (SEQ ID NO: 863),EVDPIGHLYKITNSRPPCVEVDPIGHLY (SEQ ID NO: 864) amino acids 271-279 ofMAGE-3 KITNSRPPCVFLWGPRALV (SEQ ID NO: 865),FLWGPRALVKITNSRPPCVFLWGPRALV (SEQ ID NO: 866) amino acids 167-176 ofMAGE-3 KITNSRPPCVMEVDPIGHLY (SEQ ID NO: 867),MEVDPIGHLYKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 868) amino acids 2-10 of BAGEKITNSRPPCVAARAVFLAL (SEQ ID NO: 869), AARAVFLALKITNSRPPCVAARAVFLAL (SEQID NO: 870) amino acids 9-16 of GAGE-1,2 KITNSRPPCVYRPRPRRY (SEQ ID NO:871), YRPRPRRYKITNSRPPCVYRPRPRRY (SEQ ID NO: 872) amino acids 11-20 ofRAGE KITNSRPPCVSPSSNRIRNT (SEQ ID NO: 873),SPSSNRIRNTKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 874) amino acids 23-32 ofCDK4 KITNSRPPCVACDPHSGHFV (SEQ ID NO: 875),ACDPHSGHFVKITNSRPPCVACDPHSGHFV (SEQ ID NO: 876) amino acids 29-37 ofβ-catenin KITNSRPPCVSYLDSGIHF (SEQ ID NO: 877),SYLDSGIHFKITNSRPPCVSYLDSGIHF (SEQ ID NO: 878) amino acids 1-9 oftyrosinase KITNSRPPCVMLLAVLYCL (SEQ ID NO: 879),MLLAVLYCLKITNSRPPCVMLLAVLYCL (SEQ ID NO: 880) amino acids 206-214 oftyrosinase KITNSRPPCVAFLPWHRLF (SEQ ID NO: 881),AFLPWHRLFKITNSRPPCVAFLPWHRLF (SEQ ID NO: 882) amino acids 56-70 oftyrosinase KITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 883),QNILLSNAPLGPQFPKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 884) amino acids448-462 of tyrosinase KITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 885),DYSYLQDSDPDSFQDKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 886) amino acids32-40 of Melan-A^(MART-1) KITNSRPPCVJLTVILGVL (SEQ ID NO: 887),JLTVILGVLKITNSRPPCVJLTVILGVL (SEQ ID NO: 888) amino acids 154-162 ofgp100^(Pme117) KITNSRPPCVKTWGQYWQV (SEQ ID NO: 889),KTWGQYWQVKITNSRPPCVKTWGQYWQV (SEQ ID NO: 890) amino acids 209-217 ofgp100^(Pme117) KITNSRPPCVITDQVPFSV (SEQ ID NO: 891),ITDQVPFSVKITNSRPPCVITDQVPFSV (SEQ ID NO: 892) amino acids 280-288 ofgp100^(Pme117) KITNSRPPCVYLEPGPVTA (SEQ ID NO: 893),YLEPGPVTAKITNSRPPCVYLEPGPVTA (SEQ ID NO: 894) amino acids 457-466 ofgp100^(Pme117) KITNSRPPCVLLDGTATLRL (SEQ ID NO: 895),LLDGTATLRLKITNSRPPCVLLDGTATLRL (SEQ ID NO: 896) amino acids 476-485 ofgp100^(Pme117) KITNSRPPCVVLYRYGSFSV (SEQ ID NO: 897),VLYRYGSFSVKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 898) amino acids 301-309 ofPRAME KITNSRPPCVLYVDSLFFL (SEQ ID NO: 899), LYVDSLFFLKITNSRPPCVLYVDSLFFL(SEQ ID NO: 900) amino acids 292-303 of MAGE-6 KITNSRPPCVKISGGPRISYPL(SEQ ID NO: 901), KISGGPRISYPLKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 902)amino acids 157-167 of NY-ESO-1 KITNSRPPCVSLLMWITQCFL (SEQ ID NO: 903),SLLMWITQCFLKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 904) amino acids 157-165 ofNY-ESO-1 KITNSRPPCVSLLMWITQC (SEQ ID NO: 905),SLLMWITQCKITNSRPPCVSLLMWITQC (SEQ ID NO: 906) amino acids 155-163 ofNY-ESO-1 KITNSRPPCVQLSLLMWIT (SEQ ID NO: 907),QLSLLMWITKITNSRPPCVQLSLLMWIT (SEQ ID NO: 908) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFKITNSRPPCVTSYVKVLHHMVKISG (SEQ ID NO: 909)amino acids 281-295 of MAGE-3 I105-V113 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 ITNSRPPCVEADPTGHSY (SEQ ID NO: 910), ITNSRPPCVEADPTGHSYITNSRPPCVEADPTGHSY (SEQ ID NO: 911) amino acids 230-238 ofMAGE-1 ITNSRPPCVSAYGEPRKL (SEQ ID NO: 912), SAYGEPRKLITNSRPPCVSAYGEPRKL(SEQ ID NO: 913) amino acids 168-176 of MAGE-3 ITNSRPPCVEVDPIGHLY (SEQID NO: 914), EVDPIGHLYITNSRPPCVEVDPIGHLY (SEQ ID NO: 915) amino acids271-279 of MAGE-3 ITNSRPPCVFLWGPRALV (SEQ ID NO: 916),FLWGPRALVITNSRPPCVFLWGPRALV (SEQ ID NO: 917) amino acids 167-176 ofMAGE-3 ITNSRPPCVMEVDPIGHLY (SEQ ID NO: 918),MEVDPIGHLYITNSRPPCVMEVDPIGHLY (SEQ ID NO: 919) amino acids 2-10 of BAGEITNSRPPCVAARAVFLAL (SEQ ID NO: 920), AARAVFLALITNSRPPCVAARAVFLAL (SEQ IDNO: 921) amino acids 9-16 of GAGE-1,2 ITNSRPPCVYRPRPRRY (SEQ ID NO:922), YRPRPRRYITNSRPPCVYRPRPRRY (SEQ ID NO: 923) amino acids 11-20 ofRAGE ITNSRPPCVSPSSNRIRNT (SEQ ID NO: 924), SPSSNRIRNTITNSRPPCVSPSSNRIRNT(SEQ ID NO: 925) amino acids 23-32 of CDK4 ITNSRPPCVACDPHSGHFV (SEQ IDNO: 926), ACDPHSGHFVITNSRPPCVACDPHSGHFV (SEQ ID NO: 927) amino acids29-37 of β-catenin ITNSRPPCVSYLDSGIHF (SEQ ID NO: 928),SYLDSGIHFITNSRPPCVSYLDSGIHF (SEQ ID NO: 929) amino acids 1-9 oftyrosinase ITNSRPPCVMLLAVLYCL (SEQ ID NO: 930),MLLAVLYCLITNSRPPCVMLLAVLYCL (SEQ ID NO: 931) amino acids 206-214 oftyrosinase ITNSRPPCVAFLPWHRLF (SEQ ID NO: 932),AFLPWHRLFITNSRPPCVAFLPWHRLF (SEQ ID NO: 933) amino acids 56-70 oftyrosinase ITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 934),QNILLSNAPLGPQFPITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 935) amino acids448-462 of tyrosinase ITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 936),DYSYLQDSDPDSFQDITNSRPPCVDSYLQDSDPDSFQD (SEQ ID NO: 937) amino acids32-40 of Melan-A^(MART-1) ITNSRPPCVJLTVILGVL (SEQ ID NO: 938),JLTVILGVLITNSRPPCVJLTVILGVL (SEQ ID NO: 939) amino acids 154-162 ofgp100^(Pme117) ITNSRPPCVKTWGQYWQV (SEQ ID NO: 940),KTWGQYWQVITNSRPPCVKTWGQYWQV (SEQ ID NO: 941) amino acids 209-217 ofgp100^(Pme117) ITNSRPPCVITDQVPFSV (SEQ ID NO: 942),ITDQVPFSVITNSRPPCVITDQVPFSV (SEQ ID NO: 943) amino acids 280-288 ofgp100^(Pme117) ITNSRPPCVYLEPGPVTA (SEQ ID NO: 944),YLEPGPVTAITNSRPPCVYLEPGPVTA (SEQ ID NO: 945) amino acids 457-466 ofgp100^(Pme117) ITNSRPPCVLLDGTATLRL (SEQ ID NO: 946),LLDGTATLRLITNSRPPCVLLDGTATLRL (SEQ ID NO: 947) amino acids 476-485 ofgp100^(Pme117) ITNSRPPCVVLYRYGSFSV (SEQ ID NO: 948),VLYRYGSFSVITNSRPPCVVLYRYGSFSV (SEQ ID NO: 949) amino acids 301-309 ofPRAME ITNSRPPCVLYVDSLFFL (SEQ ID NO: 950), LYVDSLFFLITNSRPPCVLYVDSLFFL(SEQ ID NO: 951) amino acids 292-303 of MAGE-6 ITNSRPPCVKISGGPRISYPL(SEQ ID NO: 952), KISGGPRISYPLITNSRPPCVKISGGPRISYPL (SEQ ID NO: 953)amino acids 157-167 of NY-ESO-1 ITNSRPPCVSLLMWITQCFL (SEQ ID NO: 954),SLLMWITQCFLITNSRPPCVSLLMWITQCFL (SEQ ID NO: 955) amino acids 157-165 ofNY-ESO-1 ITNSRPPCVSLLMWITQC (SEQ ID NO: 956),SLLMWITQCITNSRPPCVSLLMWITQC (SEQ ID NO: 957) amino acids 155-163 ofNY-ESO-1 ITNSRPPCVQLSLLMWIT (SEQ ID NO: 958),QLSLLMWITITNSRPPCVQLSLLMWIT (SEQ ID NO: 959) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFITNSRPPCVTSYVKVLHHMVKISG (SEQ ID NO: 960)amino acids 281-295 of MAGE-3 T101-V113 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 TLEKITNSRPPCVEADPTGHSY (SEQ ID NO: 961), TLEKITNSRPPCVEADPTGHSYTLEKITNSRPPCVEADPTGHSY (SEQ ID NO: 962) amino acids 230-238 ofMAGE-1 TLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 963),SAYGEPRKLTLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 964) amino acids 168-176 ofMAGE-3 TLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 965), EVDPIGHLYTLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 966) amino acids 271-279 of MAGE-3TLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 967), FLWGPRALVTLEKITNSRPPCVFLWGPRALV(SEQ ID NO: 968) amino acids 167-176 of MAGE-3 TLEKITNSRPPCVMEVDPIGHLY(SEQ ID NO: 969), MEVDPIGHLYTLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 970)amino acids 2-10 of BAGE TLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 971),AARAVFLALTLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 972) amino acids 9-16 ofGAGE-1,2 TLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 973),YRPRPRRYTLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 974) amino acids 11-20 of RAGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 975),SPSSNRIRNTTLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 976) amino acids 23-32 ofCDK4 TLEKITNSRPPCVACDPHSGHFV (SEQ ID NO: 977),ACDPHSGHFVTLEKITNSRPPCVACDPHSGHFV (SEQ ID NO: 978) amino acids 29-37 ofβ-catenin TLEKITNSRPPCVSYLDSGIHF (SEQ ID NO: 979),SYLDSGIHFTLEKITNSRPPCVSYLDSGIHF (SEQ ID NO: 980) amino acids 1-9 oftyrosinase TLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 981),MLLAVLYCLTLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 982) amino acids 206-214 oftyrosinase TLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 983),AFLPWHRLFTLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 984) amino acids 56-70 oftyrosinase TLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 985),QNILLSNAPLGPQFPTLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 986) amino acids448-462 of tyrosinase TLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 987),DYSYLQDSDPDSFQDTLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 988) amino acids32-40 of Melan-A^(MART-1) TLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 989),JLTVILGVLTLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 990) amino acids 154-162 ofgp100^(Pme117) TLEKITNSRPPCVKTWGQYWQV (SEQ ID NO: 991),KTWGQYWQVTLEKITNSRPPCVKTWGQYWQV (SEQ ID NO: 992) amino acids 209-217 ofgp100^(Pme117) TLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 993),ITDQVPFSVTLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 994) amino acids 280-288 ofgp100^(Pme117) TLEKITNSRPPCVYLEPGPVTA (SEQ ID NO: 995),YLEPGPVTATLEKITNSRPPCVYLEPGPVTA (SEQ ID NO: 996) amino acids 457-466 ofgp100^(Pme117) TLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 997),LLDGTATLRLTLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 998) amino acids 476-485of gp100^(Pme117) TLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 999),VLYRYGSFSVTLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1000) amino acids 301-309of PRAME TLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1001),LYVDSLFFLTLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1002) amino acids 292-303 ofMAGE-6 TLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1003),KISGGPRISYPLTLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1004) amino acids157-167 of NY-ESO-1 TLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1005),SLLMWITQCFLTLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1006) amino acids157-165 of NY-ESO-1 TLEKITNSRPPCVSLLMWITQC (SEQ ID NO: 1007),SLLMWITQCTLEKITNSRPPCVSLLMWITQC (SEQ ID NO: 1008) amino acids 155-163 ofNY-ESO-1 TLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1009),QLSLLMWITTLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1010) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVF TLEKITNSRPPCVTSYVKVLHHMVKISG (SEQ ID NO:1011) amino acids 281-295 of MAGE-3 193-V113 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 IRRASNGETLEKITNSRPPCVEADPTGHSY (SEQ ID NO: 1012),IRRASNGETLEKITNSRPPCV EADPTGHSYIRRASNGETLEKITNSRPPCVEADPTGHSY (SEQ IDNO: 1013) amino acids 230-238 of MAGE-1 IRRASNGETLEKITNSRPPCVSAYGEPRKL(SEQ ID NO: 1014), SAYGEPRKLIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO:1015) amino acids 168-176 of MAGE-3 IRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQID NO: 1016), EVDPIGHLYIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 1017)amino acids 271-279 of MAGE-3 IRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO:1018), FLWGPRALVIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 1019) aminoacids 167-176 of MAGE-3 IRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO:1020), MEVDPIGHLYIRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 1021) aminoacids 2-10 of BAGE IRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1022),AARAVFLALIRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1023) amino acids9-16 of GAGE-1,2 IRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1024),YRPRPRRYIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1025) amino acids11-20 of RAGE IRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 1026),SPSSNRIRNTIRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 1027) amino acids23-32 of CDK4 IRRASNGETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1028),ARDPHSGHFVIRRASNGETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1029) amino acids29-37 of β-catenin IRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1030),SYLDSGIHSIRRASNGETLEKITNSRPPCV SYLDSGIHS (SEQ ID NO: 1031) amino acids1-9 of tyrosinase IRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1032),MLLAVLYCLIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1033) amino acids206-214 of tyrosinase IRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 1034),AFLPWHRLFIRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 1035) amino acids56-70 of tyrosinase IRRASNGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO:1036), QNILLSNAPLGPQFPIRRASNGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO:1037) amino acids 448-462 of tyrosinaseIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 1038),DYSYLQDSDPDSFQDIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 1039)amino acids 32-40 of Melan-A^(MART-1) IRRASNGETLEKITNSRPPCVJLTVILGVL(SEQ ID NO: 1040) JLTVILGVLIRRASNGETLEKITNSRPPCV JLTVILGVL (SEQ ID NO:1041) amino acids 154-162 of gp100^(Pme117)IRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO: 1042),KTWGQYWQVIRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO: 1043) amino acids209-217 of gp100^(Pme117) IRRASNGETLEKITNSRPPCVILITDQVPFSV (SEQ ID NO:1044), ITDQVPFSVIRRASNGETLEKITNSRPPCVILITDQVPFSV (SEQ ID NO: 1045) aminoacids 280-288 of gp100^(Pme117) IRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQ IDNO: 1046), YLEPGPVTAIRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQ ID NO: 1047)amino acids 457-466 of gp100^(Pme117) IRRASNGETLEKITNSRPPCVLLDGTATLRL(SEQ ID NO: 1048), LLDGTATLRLIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO:1049) amino acids 476-485 of gp100^(Pme117)IRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1050),VLYRYGSFSVIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1051) amino acids301-309 of PRAME IRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1052),LYVDSLFFLIRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1053) amino acids292-303 of MAGE-6 IRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1054),KISGGPRISYPLIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1055) aminoacids 157-167 of NY-ESO-1 IRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO:1056), SLLMWITQCFLIRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1057)amino acids 157-165 of NY-ESO-1 IRRASNGETLEKITNSRPPCVSLLMWITQC (SEQ IDNO: 1058), SLLMWITQCIRRASNGETLEKITNSRPPCVSLLMWITQC (SEQ ID NO: 1059)amino acids 155-163 of NY-ESO-1 IRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ IDNO: 1060), QLSLLMWITIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1061)amino acids 157-170 of NY-ESO-1 andSLLMWITQCFLPVFIRRASNGETLEKITNSRPPCVTSYVKVLHHMVKISG (SEQ ID NO: 1062)amino acids 281-295 of MAGE-3 D88-V113 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 DLIEAIRRASNGETLEKITNSRPPCVEADPTGHSY (SEQ ID NO: 1063),DLIEAIRRASNGETLEKITN EADPTGHSYDLIEAIRRASNGETLEKITNSRPPCVEADPTGHSY (SEQID NO: 1064) SRPPCV amino acids 230-238 of MAGE-1DLIEAIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1065),SAYGEPRKLDLIEAIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1066) aminoacids 168-176 of MAGE-3 DLIEAIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO:1067), EVDPIGHLYDLIEAIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 1068)amino acids 271-279 of MAGE-3 DLIEAIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQID NO: 1069), FLWGPRALVDLIEAIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO:1070) amino acids 167-176 of MAGE-3 DLIEAIRRASNGETLEKITNSRPPCVMEVDPIGHLY(SEQ ID NO: 1071), MEVDPIGHLYDLIEAIRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQID NO: 1072) amino acids 2-10 of BAGEDLIEAIRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1073),AARAVFLALDLIEAIRRASNGETLEKITNSRPPCVAAR4VFLAL (SEQ ID NO: 1074) aminoacids 9-16 of GAGE-1,2 DLIEAIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO:1075), YRPRPRRYDLIEAIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1076)amino acids 11-20 of RAGE DLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ IDNO: 1077), SPSSNRIRNTDLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO:1078) amino acids 23-32 of CDK4 DLIEAIRRASNGETLEKITNSRPPCVARDPHSGHFV(SEQ ID NO: 1079), ARDPHSGHFVDLIEAIRRASNGETLEKITNSRPPCVARDPHSGHFV (SEQID NO: 1080) amino acids 29-37 of β-cateninDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1081),SYLDSGIHSDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1082) aminoacids 1-9 of tyrosinase DLIEAIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO:1083), MLLAVLYCLDLIEAIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1084)amino acids 206-214 of tyrosinase DLIEAIRRASNGETLEKITNSRPPCVAFLPWHRLF(SEQ ID NO: 1085), AFLPWHRLFDLIEAIRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQ IDNO: 1086) amino acids 56-70 of tyrosinaseDLIEAIRRASNGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 1087),QNILLSNAPLGPQFPDLIEAIRRASNGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO:1088) amino acids 448-462 of tyrosinaseDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 1089),DYSYLQDSDPDSFQDDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO:1090) amino acids 32-40 of Melan-A^(MART-1)DLIEAIRRASNGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 1091),JLTVILGVLDLIEAIRRASNGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 1092) aminoacids 154-162 of gp100^(Pme117) DLIEAIRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQID NO: 1093), KTWGQYWQVDLIEAIRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO:1094) amino acids 209-217 of gp100^(Pme117)DLIEAIRRASNGETLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 1095),ITDQVPFSVDLIEAIRRASNGETLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 1096) aminoacids 280-288 of gp100^(Pme117) DLIEAIRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQID NO: 1097), YLEPGPVTADLIEAIRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQ ID NO:1098) amino acids 457-466 of gp100^(Pme117)DLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1099),LLDGTATLRLDLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1100) aminoacids 476-485 of gp100^(Pme117) DLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV(SEQ ID NO: 1101), VLYRYGSFSVDLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQID NO: 1102) amino acids 301-309 of PRAMEDLIEAIRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1103),LYVDSLFFLDLIEAIRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1104) aminoacids 292-303 of MAGE-6 DLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ IDNO: 1105), KISGGPRISYPLDLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ IDNO: 1106) amino acids 157-167 of NY-ESO-1DLIEAIRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1107),SLLMWITQCFLDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1108) aminoacids 157-165 of NY-ESO-1 DLIEAIRRASNGETLEKITNSRPPCVSLLMWITQC (SEQ IDNO: 1109), SLLMWITQCDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQC (SEQ ID NO:1110) amino acids 155-163 of NY-ESO-1DLIEAIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1111),QLSLLMWITDLIEAIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1112) aminoacids 157-170 of NY-ESO-1 andSLLMWITQCFLPVFDLIEAIRRASNGETLEKITNSRPPCVTSYVKVLHHMVKISG (SEQ ID NO:1113) amino acids 281-295 of MAGE-3 P84-V113 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 PYEKDLIEAIRRASNGETLEKITNSRPPCVEADPTGHSY (SEQ ID NO:1114), PYEKDLIEAIRRASNGETLEEADPTGHSYPYEKDLIEAIRRASNGETLEKITNSRPPCVEADPTGHSY (SEQ ID NO: 1115)KITNSRPPCV amino acids 230-238 of MAGE-1PYEKDLIEAIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1116),SAYGEPRKLPYEKDLIEAIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1117) aminoacids 168-176 of MAGE-3 PYEKDLIEAIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ IDNO: 1118), EVDPIGHLYPYEKDLIEAIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO:1119) amino acids 271-279 of MAGE-3PYEKDLIEAIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 1120),FLWGPRALVPYEKDLIEAIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 1121) aminoacids 167-176 of MAGE-3 PYEKDLIEAIRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQ IDNO: 1122), MEVDPIGHLYPYEKDLIEAIRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQ IDNO: 1123) amino acids 2-10 of BAGEPYEKDLIEAIRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1124),AARAVFLALPYEKDLIEAIRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1125) aminoacids 9-16 of GAGE-1,2 PYEKDLIEAIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ IDNO: 1126), YRPRPRRYPYEKDLIEAIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO:1127) amino acids 11-20 of RAGE PYEKDLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT(SEQ ID NO: 1128), SPSSNRIRNTPYEKDLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT(SEQ ID NO: 1129) amino acids 23-32 of CDK4PYEKDLIEAIRRASNGETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1130),ARDPHSGHFVPYEKDLIEAIRRASNGETLEKITNSRPPCV ARDPHSGHFV (SEQ ID NO: 1131)amino acids 29-37 of β-catenin PYEKDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS(SEQ ID NO: 1132), SYLDSGIHSPYEKDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQID NO: 1133) amino acids 1-9 of tyrosinasePYEKDLIEAIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1134),MLLAVLYCLPYEKDLIEAIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1135) aminoacids 206-214 of tyrosinase PYEKDLIEAIRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQID NO: 1136), AFLPWHRLFPYEKDLIEAIRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQ IDNO: 1137) amino acids 56-70 of tyrosinasePYEKDLIEAIRRASNGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 1138),QNILLSNAPLGPQFPPYEKDLIEAIRRASNGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO:1139) amino acids 448-462 of tyrosinasePYEKDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 1140),DYSYLQDSDPDSFQDPYEKDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO:1141) amino acids 32-40 of Melan-A^(MART-1)PYEKDLIEAIRRASNGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 1142),JLTVILGVLPYEKDLIEAIRRASNGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 1143) aminoacids 154-162 of gp100^(Pme117) PYEKDLIEAIRRASNGETLEKITNSRPPCVKTWGQYWQV(SEQ ID NO: 1144), KTWGQYWQVPYEKDLIEAIRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQID NO: 1145) amino acids 209-217 of gp100^(Pme117)PYEKDLIEAIRRASNGETLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 1146),ITDQVPFSVPYEKDLIEAIRRASNGETLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 1147) aminoacids 280-288 of gp100^(Pme117) PYEKDLIEAIRRASNGETLEKITNSRPPCVYLEPGPVTA(SEQ ID NO: 1148), YLEPGPVTAPYEKDLIEAIRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQID NO: 1149) amino acids 457-466 of gp100^(Pme117)PYEKDLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1150),LLDGTATLRLPYEKDLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1151)amino acids 476-485 of gp100^(Pme117)PYEKDLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1152),VLYRYGSFSVPYEKDLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1153)amino acids 301-309 of PRAME PYEKDLIEAIRRASNGETLEKITNSRPPCVLYVDSLFFL(SEQ ID NO: 1154), LYVDSLFFLPYEKDLIEAIRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQID NO: 1155) amino acids 292-303 of MAGE-6PYEKDLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1156),KISGGPRISYPLPYEKDLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1157)amino acids 157-167 of NY-ESO-1PYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1158),SLLMWITQCFLPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1159)amino acids 157-165 of NY-ESO-1 PYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQC(SEQ ID NO: 1160), SLLMWITQCPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQC (SEQID NO: 1161) amino acids 155-163 of NY-ESO-1PYEKDLIEAIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1162),QLSLLMWITPYEKDLIEAIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1163) aminoacids 157-170 of NY-ESO-1 andSLLMWITQCFLPVFPYEKDLIEAIRRASNGETLEKITNSRPPCVTSYVKVLHHMVKISG (SEQ ID NO:1164) amino acids 281-295 of MAGE-3 K77-V113 of SEQ ID NO: 2 amino acids161-169 of MAGE-1 KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV EADPTGHSY (SEQID NO: 1165), KLENGGFPYEKDLIEAIRREADPTGHSYKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV EADPTGHSY (SEQ ID NO:1166) ASNGETLEKITNSRPPCV amino acids 230-238 of MAGE-1KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1167),SAYGEPRKLKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO:1168) amino acids 168-176 of MAGE-3KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 1169),EVDPIGHLYKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO:1170) amino acids 271-279 of MAGE-3KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 1171),FLWGPRALVKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO:1172) amino acids 167-176 of MAGE-3KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 1173),MEVDPIGHLYKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO:1174) amino acids 2-10 of BAGEKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1175),AARAVFLALKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO:1176) amino acids 9-16 of GAGE-1,2KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1177),YRPRPRRYKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1178)amino acids 11-20 of RAGEKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 1179),SPSSNRIRNTKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO:1180) amino acids 23-32 of CDK4KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1181),ARDPHSGHFVKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO:1182 amino acids 29-37 of β-cateninKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1183,SYLDSGIHSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO:1184) amino acids 1-9 of tyrosinaseKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1185),MLLAVLYCLKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO:1186) amino acids 206-214 of tyrosinaseKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 1187),AFLPWHRLFKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO:1188) amino acids 56-70 of tyrosinaseKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 1189,QNILLSNAPLGPQFPKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQNILLSNAPL GPQFP(SEQ ID NO: 1190) amino acids 448-462 of tyrosinaseKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 1191),DYSYLQDSDPDSFQDKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDS DPDSFQD(SEQ ID NO: 1192) amino acids 32-40 of Melan-A^(MART-1)KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 1193),JLTVILGVLKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 1194amino acids 154-162 of gp100^(Pme117)KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO: 1195),KTWGQYWQVKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO:1196) amino acids 209-217 of gp100^(Pme117)KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVILITDQVPFSV (SEQ ID NO: 1197),ITDQVPFSVKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVILITDQVPFSV (SEQ ID NO:1198) amino acids 280-288 of gp100^(Pme117)KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQ ID NO: 1199),YLEPGPVTAKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQ ID NO:1200) amino acids 457-466 of gp100^(Pme117)KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1201),LLDGTATLRLKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO:1202) amino acids 476-485 of gp100^(Pme117)KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1203),VLYRYGSFSVKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO:1204) amino acids 301-309 of PRAMEKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1205),LYVDSLFFLKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO:1206) amino acids 292-303 of MAGE-6KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1207),KISGGPRISYPLKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ IDNO: 1208) amino acids 157-167 of NY-ESO-1KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1209),SLLMWITQCFLKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO:1210) amino acids 157-165 of NY-ESO-1KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQC (SEQ ID NO: 1211),SLLMWITQCKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQC (SEQ ID NO:1212) amino acids 155-163 of NY-ESO-1KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1213),QLSLLMWITKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1214amino acids 157-170 of NY-ESO-1 andSLLMWITQCFLPVFKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVTSYVKVLHH amino acids281-295 of MAGE-3 MVKISG (SEQ ID NO: 1215) Q72-V113 of SEQ ID NO: 2amino acids 161-169 of MAGE-1QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEADPTGHSY (SEQ ID NO: 1216),QLVFSKLENGGFPYEKDLIEADPTGHSYQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEADPTGHSY (SEQ ID NO:1217) EAIRRASNGETLEKITNSRP PCV amino acids 230-238 of MAGE-1QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1218),SAYGEPRKLQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO:1219) amino acids 168-176 of MAGE-3QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 1220),EVDPIGHLYQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO:1221) amino acids 271-279 of MAGE-3QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 1222)FLWGPRALVQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO:1223) amino acids 167-176 of MAGE-3QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 1224),MEVDPIGHLYQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMEVDPIGH LY (SEQ IDNO: 1225) amino acids 2-10 of BAGEQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1226),AARAVFLALQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO:1227) amino acids 9-16 of GAGE-1,2QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1228),YRPRPRRYQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO:1229) amino acids 11-20 of RAGEQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 1230),SPSSNRIRNTQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ IDNO: 1231) amino acids 23-32 of CDK4QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1232),ARDPHSGHFVQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVARDPHSGH FV (SEQ IDNO: 1233) amino acids 29-37 of β-cateninQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1234),SYLDSGIHSQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO:1235) amino acids 1-9 of tyrosinaseQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1236),MLLAVLYCLQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO:1237) amino acids 206-214 of tyrosinaseQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 1238),AFLPWHRLFQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO:1239) amino acids 56-70 of tyrosinaseQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO:1240), QNILLSNAPLGPQFPQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 1241) amino acids 448-462 of tyrosinaseQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO:1242), DYSYLQDSDPDSFQDQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 1243) amino acids 32-40 of Melan-A^(MART-1)QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 1244),JLTVILGVLQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO:1245) amino acids 154-162 of gp100^(Pme117)QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO: 1246),KTWGQYWQVQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKTWGQYW QV (SEQ IDNO: 1247) amino acids 209-217 of gp100^(Pme117)QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 1248),ITDQVPFSVQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVITDQVPFSV (SEQ ID NO:1249) amino acids 280-288 of gp100^(Pme117)QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQ ID NO: 1250),YLEPGPVTAQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQ ID NO:1251) amino acids 457-466 of gp100^(Pme117)QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1252),LLDGTATLRLQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ IDNO: 1253) amino acids 476-485 of gp100^(Pme117)QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1254),VLYRYGSFSVQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ IDNO: 1255) amino acids 301-309 of PRAMEQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1256),LYVDSLFFLQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO:1257) amino acids 292-303 of MAGE-6QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO:1258), KISGGPRISYPLQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1259) amino acids 157-167 of NY-ESO-1QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1260),SLLMWITQCFLQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQ CFL (SEQID NO: 1261) amino acids 157-165 of NY-ESO-1QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQC (SEQ ID NO: 1262),SLLMWITQCQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQC (SEQ ID NO:1263) amino acids 155-163 of NY-ESO-1QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1264),QLSLLMWITQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO:1265) amino acids 157-170 of NY-ESO-1 andSLLMWITQCFLPVFQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVTSYVK aminoacids 281-295 of MAGE-3 VLHHMVKISG (SEQ ID NO: 1266) F65 to V113 of SEQID NO: 2 amino acids 161-169 of MAGE-1FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEADPTGHSY (SEQ ID NO:1267), FEIEINGQLVFSKLENGGFPEADPTGHSYFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEADYEKDLIEAIRRASNGETLE PTGHSY (SEQ ID NO: 1268) KITNSRPPCV amino acids230-238 of MAGE-1FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO:1269), SAYGEPRKLFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1270) amino acids 168-176 of MAGE-3FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO:1271), EVDPIGHLYFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 1272) amino acids 271-279 of MAGE-3FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO:1273), FLWGPRALVFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 1274) amino acids 167-176 of MAGE-3FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO:1275), MEVDPIGHLYFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 1276) amino acids 2-10 of BAGEFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO:1277), AARAVFLALFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1278) amino acids 9-16 of GAGE-1,2FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO:1279), YRPRPRRYFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1280) amino acids 11-20 of RAGEFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO:1281), SPSSNRIRNTFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 1282) amino acids 23-32 of CDK4FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO:1283), ARDPHSGHFVFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1284) amino acids 29-37 of β-cateninFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO:1285), SYLDSGIHSFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1286) amino acids 1-9 of tyrosinaseFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO:1287), MLLAVLYCLFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1288) amino acids 206-214 of tyrosinaseFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO:1289), AFLPWHRLFFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 1290) amino acids 56-70 of tyrosinaseFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQNILLSNAPLGPQ FP (SEQID NO: 1291),QNILLSNAPLGPQFPFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPP CVQNILLSNAPLGPQFP (SEQ ID NO: 1292) amino acids 448-462 of tyrosinaseFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDSDPDS FQD (SEQID NO: 1293),DYSYLQDSDPDSFQDFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRP PCVDYSYLQDSDPDSFQD (SEQ ID NO: 1294) amino acids 32-40 of Melan-A^(MART-1)FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO:1295), JLTVILGVLFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 1296) amino acids 154-162 of gp100^(Pme117)FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO:1297), KTWGQYWQVFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO: 1298 amino acids 209-217 of gp100^(Pme117)FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVITDQVPFSV (SEQ ID NO:1299), ITDQVPFSVFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 1300) amino acids 280-288 of gp100^(Pme117)FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQ ID NO:1301), YLEPGPVTAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYLEPGPVTA (SEQ ID NO: 1302) amino acids 457-466 of gp100^(Pme117)FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO:1303), LLDGTATLRLFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1304) amino acids 476-485 of gp100^(Pme117)FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO:1305), VLYRYGSFSVFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1306) amino acids 301-309 of PRAMEFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO:1307), LYVDSLFFLFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1308) amino acids 292-303 of MAGE-6FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ IDNO: 1309), KISGGPRISYPLFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1310) amino acids 157-167 of NY-ESO-1FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVILSLLMWITQCFL (SEQ IDNO: 1311), SLLMWITQCFLFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1312) amino acids 157-165 of NY-ESO-1FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQC (SEQ ID NO:1313), SLLMWITQCFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQC (SEQ ID NO: 1314) amino acids 155-163 of NY-ESO-1FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO:1315), QLSLLMWITFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1316) amino acids 157-170 of NY-ESO-1 andSLLMWITQCFLPVFFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPP aminoacids 281-295 of MAGE-3 CVTSYVKVLHHMVKISG (SEQ ID NO: 1317) L59-V113 ofSEQ ID NO: 2 amino acids 161-169 of MAGE-1LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEADPTLGGTGAFEIEINGQLVFSKL GHSY (SEQ ID NO: 1318), ENGGFPYEKDLIEAIRRASEADPTGHSYLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRNGETLEKITNSRPPCV PPCV EADPTGHSY (SEQ ID NO: 1319) amino acids 230-238 ofMAGE-1 LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSAYGE PRKL(SEQ ID NO: 1320),SAYGEPRKLLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSR PPCVSAYGEPRKL (SEQ ID NO: 1321) amino acids 168-176 of MAGE-3LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVEVDPIG HLY (SEQID NO: 1322),EVDPIGHLYLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRP PCVEVDPIGHLY (SEQ ID NO: 1323) amino acids 271-279 of MAGE-3LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVFLWGP RALV (SEQID NO: 1324),FLWGPRALVLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSR PPCVFLWGPRALV (SEQ ID NO: 1325) amino acids 167-176 of MAGE-3LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMEVDP IGHLY (SEQID NO: 1326),MEVDPIGHLYLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 1327) amino acids 2-10 of BAGELGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAARAV FLAL (SEQID NO: 1328),AARAVFLALLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSR PPCVAARAVFLAL (SEQ ID NO: 1329) amino acids 9-16 of GAGE-1,2LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYRPRP RRY (SEQ IDNO: 1330), YRPRPRRYLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV YRPRPRRY (SEQ ID NO: 1331) amino acids 11-20 of RAGELGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSPSSNR IRNT (SEQID NO: 1332),SPSSNRIRNTLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 1333) amino acids 23-32 of CDK4LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVARDPH SGHFV (SEQID NO: 1334),ARDPHSGHFVLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1335) amino acids 29-37 of β-cateninLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSYLDS GIHS (SEQID NO: 1336),SYLDSGIHSLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1337) amino acids 1-9 of tyrosinaseLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMLLAV LYCL (SEQID NO: 1338),MLLAVLYCLLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSR PPCVMLLAVLYCL (SEQ ID NO: 1339) amino acids 206-214 of tyrosinaseLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAFLPW HRLF (SEQID NO: 1340),AFLPWHRLFLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSR PPCVAFLPWHRLF (SEQ ID NO: 1341) amino acids 56-70 of tyrosinaseLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQNILLS NAPLGPQFP(SEQ ID NO: 1342),QNILLSNAPLGPQFPLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 1343) amino acids 448-462 oftyrosinase LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 1344),DYSYLQDSDPDSFQDLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 1345) amino acids 32-40 ofMelan-A^(MART-1)LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVJLTVIL GVL (SEQID NO: 1346),JLTVILGVLLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRP PCVJLTVILGVL (SEQ ID NO: 1347) amino acids 154-162 of gp100^(Pme117)LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKTWGQ YWQV (SEQID NO: 1348),KTWGQYWQVLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO: 1349) amino acids 209-217 of gp100^(Pme117)LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVITDQVP FSV (SEQID NO: 1350),ITDQVPFSVLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 1351) amino acids 280-288 of gp100^(Pme117)LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVYLEPGP VTA (SEQID NO: 1352),YLEPGPVTALGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRP PCVYLEPGPVTA (SEQ ID NO: 1353) amino acids 457-466 of gp100^(Pme117)LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLLDGT ATLRL (SEQID NO: 1354),LLDGTATLRLLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1355) amino acids 476-485 of gp100^(Pme117)LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVVLYRY GSFSV (SEQID NO: 1356),VLYRYGSFSVLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1357) amino acids 301-309 of PRAMELGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVLYVDS LFFL (SEQID NO: 1358),LYVDSLFFLLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRP PCVLYVDSLFFL (SEQ ID NO: 1359) amino acids 292-303 of MAGE-6LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKISGGP RISYPL(SEQ ID NO: 1360),KISGGPRISYPLLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1361) amino acids 157-167 of NY-ESO-1LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMW ITQCFL (SEQID NO: 1362),SLLMWITQCFLLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1363) amino acids 157-165 of NY-ESO-1LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVSLLMW ITQC (SEQID NO: 1364),SLLMWITQCLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSR PPCVSLLMWITQC (SEQ ID NO: 1365) amino acids 155-163 of NY-ESO-1LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVQLSLL MWIT (SEQID NO: 1366),QLSLLMWITLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSR PPCVQLSLLMWIT (SEQ ID NO: 1367) amino acids 157-170 of NY-ESO-1 andSLLMWITQCFLPVFLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKI aminoacids 281-295 of MAGE-3 TNSRPPCVTSYVKVLHHMVKISG (SEQ ID NO: 1368)G99-V113 of SEQ ID NO: 2 amino acids 161-169 of MAGE-1GETLEKITNSRPPCVEADPTGHSY (SEQ ID NO: 1369), GETLEKITNSRPPCVEADPTGHSYGETLEKITNSRPPCVEADPTGHSY (SEQ ID NO: 1370) amino acids 230-238of MAGE-1 GETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1371),SAYGEPRKLGETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1372) amino acids 168-176of MAGE-3 GETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 1373),EVDPIGHLYGETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 1374) amino acids 271-279of MAGE-3 GETLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 1375),FLWGPRALVGETLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 1376) amino acids 167-176of MAGE-3 GETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 1377),MEVDPIGHLYGETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 1378) amino acids 2-10of BAGE GETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1379),AARAVFLALGETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1380) amino acids 9-16 ofGAGE-1,2 GETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1381),YRPRPRRYGETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1382) amino acids 11-20 ofRAGE GETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 1383),SPSSNRIRNTGETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 1384) amino acids 23-32of CDK4 GETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1385),ARDPHSGHFVGETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1386) amino acids 29-37of β-catenin GETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1387),SYLDSGIHSGETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1388) amino acids 1-9 ofTyrosinase GETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1389),MLLAVLYCLGETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1390) amino acids 206-214of Tyrosinase GETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 1391),AFLPWHRLFGETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 1392) amino acids 56-70 ofTyrosinase GETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 1393),QNILLSNAPLGPQFPGETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 1394) aminoacids 448-462 of Tyrosinase GETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO:1395), DYSYLQDSDPDSFQDGETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 1396)amino acids 32-40 of Melan-A^(MART-1) GETLEKITNSRPPCVJLTVILGVL (SEQ IDNO: 1397), JLTVILGVLGETLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 1398) aminoacids 154-162 of gp100^(Pme117) GETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO:1399, KTWGQYWQVGETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO: 1400) amino acids209-217 of gp100^(Pme117) SVAPPPEEVITDQVPFSV (SEQ ID NO: 1401),ITDQVPFSVSVAPPPEEVITDQVPFSV (SEQ ID NO: 1402) amino acids 280-288 ofgp100^(Pme117) SVAPPPEEVYLEPGPVTA (SEQ ID NO: 1403),YLEPGPVTASVAPPPEEVYLEPGPVTA (SEQ ID NO: 1404) amino acids 457-466 ofgp100^(Pme117) GETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1405),LLDGTATLRLGETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1406) amino acids476-485 of gp100^(Pme117) GETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1407),VLYRYGSFSVGETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1408) amino acids301-309 of PRAME GETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1409),LYVDSLFFLGETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1410) amino acids 292-303of MAGE-6 GETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1411),KISGGPRISYPLGETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1412) amino acids157-167 of NY-ESO-1 GETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1413),SLLMWITQCFLGETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1414) amino acids157-165 of NY-ESO-1 GETLEKITNSRPPCVSLLMWITQC (SEQ ID NO: 1415),SLLMWITQCGETLEKITNSRPPCVSLLMWITQC (SEQ ID NO: 1416) amino acids 155-163of NY-ESO-1 GETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1417),QLSLLMWITGETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1418) amino acids 157-170of NY-ESO-1 and SLLMWITQCFLPVFGETLEKITNSRPPCVTSYVKVLHHMVKISG (SEQ ID NO:1419) amino acids 281-295 of MAGE-3 E100-V113 of SEQ ID NO: 2 aminoacids 161-169 of MAGE-1 ETLEKITNSRPPCVADPTGHSY (SEQ ID NO: 1420),ETLEKITNSRPPCV EADPTGHSYETLEKITNSRPPCVEADPTGHSY (SEQ ID NO: 1421) aminoacids 230-238 of MAGE-1 ETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1422),SAYGEPRKLETLEKITNSRPPCVSAYGEPRKL (SEQ ID NO: 1423) amino acids 168-176of MAGE-3 ETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 1424), EVDPIGHLYETLEKITNSRPPCVEVDPIGHLY (SEQ ID NO: 1425) amino acids 271-279 of MAGE-3ETLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 1426),FLWGPRALVETLEKITNSRPPCVFLWGPRALV (SEQ ID NO: 1427) amino acids 167-176of MAGE-3 ETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 1428),MEVDPIGHLYETLEKITNSRPPCVMEVDPIGHLY (SEQ ID NO: 1429) amino acids 2-10 ofBAGE ETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1430),AARAVFLALETLEKITNSRPPCVAARAVFLAL (SEQ ID NO: 1431) amino acids 9-16 ofGAGE-1,2 ETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1432),YRPRPRRYETLEKITNSRPPCVYRPRPRRY (SEQ ID NO: 1433) amino acids 11-20 ofRAGE ETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 1434),SPSSNRIRNTETLEKITNSRPPCVSPSSNRIRNT (SEQ ID NO: 1435) amino acids 23-32of CDK4 ETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1436,ARDPHSGHFVETLEKITNSRPPCVARDPHSGHFV (SEQ ID NO: 1437) amino acids 29-37of β-catenin ETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1438),SYLDSGIHSETLEKITNSRPPCVSYLDSGIHS (SEQ ID NO: 1439) amino acids 1-9 ofTyrosinase ETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1440),MLLAVLYCLETLEKITNSRPPCVMLLAVLYCL (SEQ ID NO: 1441) amino acids 206-214of Tyrosinase ETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 1442),AFLPWHRLFETLEKITNSRPPCVAFLPWHRLF (SEQ ID NO: 1443) amino acids 56-70 ofTyrosinase ETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 1444),QNILLSNAPLGPQFPETLEKITNSRPPCVQNILLSNAPLGPQFP (SEQ ID NO: 1445) aminoacids 448-462 of Tyrosinase ETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO:1446), DYSYLQDSDPDSFQDETLEKITNSRPPCVDYSYLQDSDPDSFQD (SEQ ID NO: 1447)amino acids 32-40 of Melan-A^(MART-1) ETLEKITNSRPPCVJLTVILGVL (SEQ IDNO: 1448), JLTVILGVLETLEKITNSRPPCVJLTVILGVL (SEQ ID NO: 1449) aminoacids 154-162 of gp100^(Pme117) ETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO:1450), KTWGQYWQVETLEKITNSRPPCVKTWGQYWQV (SEQ ID NO: 1451) amino acids209-217 of gp100^(Pme117) ETLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 1452),ITDQVPFSVETLEKITNSRPPCVITDQVPFSV (SEQ ID NO: 1453) amino acids 280-288of gp100^(Pme117) ETLEKITNSRPPCVYLEPGPVTA (SEQ ID NO: 1454),YLEPGPVTAETLEKITNSRPPCVYLEPGPVTA (SEQ ID NO: 1455) amino acids 457-466of gp100^(Pme117) ETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1456),LLDGTATLRLETLEKITNSRPPCVLLDGTATLRL (SEQ ID NO: 1457) amino acids 476-485of gp100^(Pme117) ETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1458),VLYRYGSFSVETLEKITNSRPPCVVLYRYGSFSV (SEQ ID NO: 1459) amino acids 301-309of PRAME ETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1460),LYVDSLFFLETLEKITNSRPPCVLYVDSLFFL (SEQ ID NO: 1461) amino acids 292-303of MAGE-6 ETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1462),KISGGPRISYPLETLEKITNSRPPCVKISGGPRISYPL (SEQ ID NO: 1463) amino acids157-167 of NY-ESO-1 ETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1464),SLLMWITQCFLETLEKITNSRPPCVSLLMWITQCFL (SEQ ID NO: 1465) amino acids157-165 of NY-ESO-1 ETLEKITNSRPPCVSLLMWITQC (SEQ ID NO: 1466),SLLMWITQCETLEKITNSRPPCVSLLMWITQC (SEQ ID NO: 1467) amino acids 155-163of NY-ESO-1 ETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1468),QLSLLMWITETLEKITNSRPPCVQLSLLMWIT (SEQ ID NO: 1469) amino acids 157-170of NY-ESO-1 and SLLMWITQCFLPVFETLEKITNSRPPCVTSYVKVLHHMVKISG (SEQ ID NO:1470) amino acids 281-295 of MAGE-3

TABLE C C35 Epitope Analog Exemplary Tumor Rejection Peptide ExemplaryPolytope Analog of S77-Y85 of SEQ ID amino acids 161-169 of MAGE-1KLENGGFPVEADPTGHSY (SEQ ID NO: 1471), NO: 2 having valine substitutedEADPTGHSYKLENGGFPVEADPTGHSY (SEQ ID NO: 1472) for tyrosine at ninthamino acid residue (KLENGGFPV, SEQ ID NO: 171) amino acids 230-238 ofMAGE-1 KLENGGFPVSAYGEPRKL (SEQ ID NO: 1473), SAYGEPRKLKLENGGFPVSAYGEPRKL(SEQ ID NO: 1474) amino acids 168-176 of MAGE-3 KLENGGFPVEVDPIGHLY (SEQID NO: 1475), EVDPIGHLYKLENGGFPVEVDPIGHLY (SEQ ID NO: 1476) amino acids271-279 of MAGE-3 KLENGGFPVFLWGPRALV (SEQ ID NO: 1477),FLWGPRALVKLENGGFPVFLWGPRALV (SEQ ID NO: 1478) amino acids 167-176 ofMAGE-3 KLENGGFPVMEVDPIGHLY (SEQ ID NO: 1479),MEVDPIGHLYKLENGGFPVMEVDPIGHLY (SEQ ID NO: 1480) amino acids 2-10 of BAGEKLENGGFPVAARAVFLAL (SEQ ID NO: 1481), AARAVFLALKLENGGFPVAARAVFLAL (SEQID NO: 1482) amino acids 9-16 of GAGE-1,2 KLENGGFPVYRPRPRRY (SEQ ID NO:1483), YRPRPRRYKLENGGFPVYRPRPRRY (SEQ ID NO: 1484) amino acids 11-20 ofRAGE KLENGGFPVSPSSNRIRNT (SEQ ID NO: 1485),SPSSNRIRNTKLENGGFPVSPSSNRIRNT (SEQ ID NO: 1486) amino acids 23-32 ofCDK4 KLENGGFPVARDPHSGHFV (SEQ ID NO: 1487),ARDPHSGHFVKLENGGFPVARDPHSGHFV (SEQ ID NO: 1488) amino acids 29-37 ofβ-catenin KLENGGFPVSYLDSGIHS (SEQ ID NO: 1489),SYLDSGIHSKLENGGFPVSYLDSGIHS (SEQ ID NO: 1490) amino acids 1-9 ofTyrosinase KLENGGFPVMLLAVLYCL (SEQ ID NO: 1500),MLLAVLYCLKLENGGFPVMLLAVLYCL (SEQ ID NO: 1501) amino acids 206-214 ofTyrosinase KLENGGFPVAFLPWHRLF (SEQ ID NO: 1502),AFLPWHRLFKLENGGFPVAFLPWHRLF (SEQ ID NO: 1503) amino acids 56-70 ofTyrosinase KLENGGFPVQNILLSNAPLGPQFP (SEQ ID NO: 1504),QNILLSNAPLGPQFPKLENGGFPVQNILLSNAPLGPQFP (SEQ ID NO: 1505) amino acids448-462 of Tyrosinase KLENGGFPVDYSYLQDSDPDSFQD (SEQ ID NO: 1506),DYSYLQDSDPDSFQDKLENGGFPVDYSYLQDSDPDSFQD (SEQ ID NO: 1507) amino acids32-40 of Melan-A^(MART-1) KLENGGFPVJLTVILGVL (SEQ ID NO: 1508),JLTVILGVLKLENGGFPVJLTVILGVL (SEQ ID NO: 1509) amino acids 154-162 ofgp100^(Pme117) KLENGGFPVKTWGQYWQV (SEQ ID NO: 1510),KTWGQYWQVKLENGGFPVKTWGQYWQV (SEQ ID NO: 1511) amino acids 209-217 ofgp100^(Pme117) KLENGGFPVITDQVPFSV (SEQ ID NO: 1512),ITDQVPFSVKLENGGFPVITDQVPFSV (SEQ ID NO: 1513) amino acids 280-288 ofgp100^(Pme117) KLENGGFPVYLEPGPVTA (SEQ ID NO: 1514),YLEPGPVTAKLENGGFPVYLEPGPVTA (SEQ ID NO: 1515) amino acids 457-466 ofgp100^(Pme117) KLENGGFPVLLDGTATLRL (SEQ ID NO: 1516),LLDGTATLRLKLENGGFPVLLDGTATLRL (SEQ ID NO: 1517) amino acids 476-485 ofgp100^(Pme117) KLENGGFPVVLYRYGSFSV (SEQ ID NO: 1518),VLYRYGSFSVKLENGGFPVVLYRYGSFSV (SEQ ID NO: 1519) amino acids 301-309 ofPRAME KLENGGFPVLYVDSLFFL (SEQ ID NO: 1520), LYVDSLFFLKLENGGFPVLYVDSLFFL(SEQ ID NO: 1521) amino acids 292-303 of MAGE-6 KLENGGFPVKISGGPRISYPL(SEQ ID NO: 1522), KISGGPRISYPLKLENGGFPVKISGGPRISYPL (SEQ ID NO: 1523)amino acids 157-167 of NY-ESO-1 KLENGGFPVSLLMWITQCFL (SEQ ID NO: 1524),SLLMWITQCFLKLENGGFPVSLLMWITQCFL (SEQ ID NO: 1525) amino acids 157-165 ofNY-ESO-1 KLENGGFPVSLLMWITQC (SEQ ID NO: 1526),SLLMWITQCKLENGGFPVSLLMWITQC (SEQ ID NO: 1527) amino acids 155-163 ofNY-ESO-1 KLENGGFPVQLSLLMWIT (SEQ ID NO: 1528),QLSLLMWITKLENGGFPVQLSLLMWIT (SEQ ID NO: 1529) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFKLENGGFPVTSYVKVLHHMVKISG (SEQ ID NO: 1530)amino acids 281-295 of MAGE-3 Analog of K104-C112 of SEQ amino acids161-169 of MAGE-1 KITNSRPPLEADPTGHSY (SEQ ID NO: 1531), ID NO: 2 havingleucine EADPTGHSYKITNSRPPLEADPTGHSY (SEQ ID NO: 1532) substituted forcysteine at the ninth amino acid residue (KITNSRPPL, SEQ ID NO: 172)amino acids 230-238 of MAGE-1 KITNSRPPLSAYGEPRKL (SEQ ID NO: 1533),SAYGEPRKLKITNSRPPLSAYGEPRKL (SEQ ID NO: 1534) amino acids 168-176 ofMAGE-3 KITNSRPPLEVDPIGHLY (SEQ ID NO: 1535), EVDPIGHLYKITNSRPPLEVDPIGHLY(SEQ ID NO: 1536) amino acids 271-279 of MAGE-3 KITNSRPPL FLWGPRALV (SEQID NO: 1537), FLWGPRALVKITNSRPPLFLWGPRALV (SEQ ID NO: 1538) amino acids167-176 of MAGE-3 KITNSRPPL MEVDPIGHLY (SEQ ID NO: 1539),MEVDPIGHLYKITNSRPPLMEVDPIGHLY (SEQ ID NO: 1540) amino acids 2-10 of BAGEKITNSRPPLAARAVFLAL (SEQ ID NO: 1541), AARAVFLALKITNSRPPLAARAVFLAL (SEQID NO: 1542) amino acids 9-16 of GAGE-1,2 KITNSRPPL YRPRPRRY (SEQ ID NO:1543), YRPRPRRYKITNSRPPLYRPRPRRY (SEQ ID NO: 1544) amino acids 11-20 ofRAGE KITNSRPPLSPSSNRIRNT (SEQ ID NO: 1545),SPSSNRIRNTKITNSRPPLSPSSNRIRNT (SEQ ID NO: 1546) amino acids 23-32 ofCDK4 KITNSRPPLARDPHSGHFV (SEQ ID NO: 1547),ARDPHSGHFVKITNSRPPLARDPHSGHFV (SEQ ID NO: 1548) amino acids 29-37 ofβ-catenin KITNSRPPLSYLDSGIHS (SEQ ID NO: 1549),SYLDSGIHSKITNSRPPLSYLDSGIHS (SEQ ID NO: 1550) amino acids 1-9 ofTyrosinase KITNSRPPL MLLAVLYCL (SEQ ID NO: 1551),MLLAVLYCLKITNSRPPLMLLAVLYCL (SEQ ID NO: 1552) amino acids 206-214 ofTyrosinase KITNSRPPLAFLPWHRLF (SEQ ID NO: 1553),AFLPWHRLFKITNSRPPLAFLPWHRLF (SEQ ID NO: 1554) amino acids 56-70 ofTyrosinase KITNSRPPLQNILLSNAPLGPQFP (SEQ ID NO: 1555),QNILLSNAPLGPQFPKITNSRPPLQNILLSNAPLGPQFP (SEQ ID NO: 1556) amino acids448-462 of Tyrosinase KITNSRPPLDYSYLQDSDPDSFQD (SEQ ID NO: 1557),DYSYLQDSDPDSFQDKITNSRPPLDYSYLQDSDPDSFQD (SEQ ID NO: 1558) amino acids32-40 of Melan-A^(MART-1) KITNSRPPLJLTVILGVL (SEQ ID NO: 1559),JLTVILGVLKITNSRPPLJLTVILGVL (SEQ ID NO: 1560) amino acids 154-162 ofgp100^(Pme117) KITNSRPPLKTWGQYWQV (SEQ ID NO: 1561),KTWGQYWQVKITNSRPPLKTWGQYWQV (SEQ ID NO: 1562) amino acids 209-217 ofgp100^(Pme117) KITNSRPPLITDQVPFSV (SEQ ID NO: 1563),ITDQVPFSVKITNSRPPLITDQVPFSV (SEQ ID NO: 1564) amino acids 280-288 ofgp100^(Pme117) KITNSRPPLYLEPGPVTA (SEQ ID NO: 1565),YLEPGPVTAKITNSRPPLYLEPGPVTA (SEQ ID NO: 1566) amino acids 457-466 ofgp100^(Pme117) KITNSRPPLLLDGTATLRL (SEQ ID NO: 1567),LLDGTATLRLKITNSRPPLLLDGTATLRL (SEQ ID NO: 1568) amino acids 476-485 ofgp100^(Pme117) KITNSRPPLVLYRYGSFSV (SEQ ID NO: 1569),VLYRYGSFSVKITNSRPPLVLYRYGSFSV (SEQ ID NO: 1570) amino acids 301-309 ofPRAME KITNSRPPLLYVDSLFFL (SEQ ID NO: 1571), LYVDSLFFLKITNSRPPLLYVDSLFFL(SEQ ID NO: 1572) amino acids 292-303 of MAGE-6 KITNSRPPLKISGGPRISYPL(SEQ ID NO: 1573), KISGGPRISYPLKITNSRPPLKISGGPRISYPL (SEQ ID NO: 1574)amino acids 157-167 of NY-ESO-1 KITNSRPPLSLLMWITQCFL (SEQ ID NO: 1575),SLLMWITQCFLKITNSRPPLSLLMWITQCFL (SEQ ID NO: 1576) amino acids 157-165 ofNY-ESO-1 KITNSRPPLSLLMWITQC (SEQ ID NO: 1577),SLLMWITQCKITNSRPPLSLLMWITQC (SEQ ID NO: 1578) amino acids 155-163 ofNY-ESO-1 KITNSRPPLQLSLLMWIT (SEQ ID NO: 1579),QLSLLMWITKITNSRPPLQLSLLMWIT (SEQ ID NO: 1580) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFKITNSRPPLTSYVKVLHHMVKISG (SEQ ID NO: 1581)amino acids 281-295 of MAGE-3 Analog of I105-V113 of SEQ amino acids161-169 of MAGE-1 ILNSRPPAVEADPTGHSY (SEQ ID NO: 1582), ID NO: 2 havingleucine EADPTGHSYILNSRPPAVEADPTGHSY (SEQ ID NO: 1583) substituted forthreonine at the second amino acid residue and alanine substituted forcysteine at the eighth amino acid residue (ILNSRPPAV, SEQ ID NO: 183)amino acids 230-238 of MAGE-1 ILNSRPPAVSAYGEPRKL (SEQ ID NO: 1584),SAYGEPRKLILNSRPPAVSAYGEPRKL (SEQ ID NO: 1585) amino acids 168-176 ofMAGE-3 ILNSRPPAVEVDPIGHLY (SEQ ID NO: 1586), EVDPIGHLYILNSRPPAVEVDPIGHLY(SEQ ID NO: 1587) amino acids 271-279 of MAGE-3 ILNSRPPAVFLWGPRALV (SEQID NO: 1588), FLWGPRALVILNSRPPAVFLWGPRALV (SEQ ID NO: 1589) amino acids167-176 of MAGE-3 ILNSRPPAVMEVDPIGHLY (SEQ ID NO: 1590),MEVDPIGHLYILNSRPPAVMEVDPIGHLY (SEQ ID NO: 1591) amino acids 2-10 of BAGEILNSRPPAVAARAVFLAL (SEQ ID NO: 1592), AARAVFLALILNSRPPAVAARAVFLAL (SEQID NO: 1593) amino acids 9-16 of GAGE-1,2 ILNSRPPAVYRPRPRRY (SEQ ID NO:1594), YRPRPRRYILNSRPPAVYRPRPRRY (SEQ ID NO: 1595) amino acids 11-20 ofRAGE ILNSRPPAVSPSSNRIRNT (SEQ ID NO: 1596),SPSSNRIRNTILNSRPPAVSPSSNRIRNT (SEQ ID NO: 1597) amino acids 23-32 ofCDK4 ILNSRPPAVARDPHSGHFV (SEQ ID NO: 1598),ARDPHSGHFVILNSRPPAVARDPHSGHFV (SEQ ID NO: 1599) amino acids 29-37 ofβ-catenin ILNSRPPAVSYLDSGIHS (SEQ ID NO: 1600), SYLDSGIHSILNSRPPAVSYLDSGIHS (SEQ ID NO: 1601) amino acids 1-9 of TyrosinaseILNSRPPAVMLLAVLYCL (SEQ ID NO: 1602), MLLAVLYCLILNSRPPAVMLLAVLYCL (SEQID NO: 1603) amino acids 206-214 of Tyrosinase ILNSRPPAVAFLPWHRLF (SEQID NO :1604), AFLPWHRLFILNSRPPAVAFLPWHRLF (SEQ ID NO: 1605) amino acids56-70 of Tyrosinase ILNSRPPAVQNILLSNAPLGPQFP (SEQ ID NO: 1606),QNILLSNAPLGPQFPILNSRPPAVQNILLSNAPLGPQFP (SEQ ID NO: 1607) amino acids448-462 of Tyrosinase ILNSRPPAVDYSYLQDSDPDSFQD (SEQ ID NO: 1608),DYSYLQDSDPDSFQDILNSRPPAVDYSYLQDSDPDSFQD (SEQ ID NO: 1609) amino acids32-40 of Melan-A^(MART-1) ILNSRPPAVJLTVILGVL (SEQ ID NO: 1610),JLTVILGVLILNSRPPAVJLTVILGVL (SEQ ID NO: 1611) amino acids 154-162 ofgp100^(Pme117) ILNSRPPAVKTWGQYWQV (SEQ ID NO: 1612),KTWGQYWQVILNSRPPAVKTWGQYWQV (SEQ ID NO: 1613) amino acids 209-217 ofgp100^(Pme117) ILNSRPPAVITDQVPFSV (SEQ ID NO: 1614),ITDQVPFSVILNSRPPAVITDQVPFSV (SEQ ID NO: 1615) amino acids 280-288 ofgp100^(Pme117) ILNSRPPAVYLEPGPVTA (SEQ ID NO: 1616),YLEPGPVTAILNSRPPAVYLEPGPVTA (SEQ ID NO: 1617) amino acids 457-466 ofgp100^(Pme117) ILNSRPPAVLLDGTATLRL (SEQ ID NO: 1618),LLDGTATLRLILNSRPPAVLLDGTATLRL (SEQ ID NO: 1619) amino acids 476-485 ofgp100^(Pme117) ILNSRPPAVVLYRYGSFSV (SEQ ID NO: 1620),VLYRYGSFSVILNSRPPAVVLYRYGSFSV (SEQ ID NO: 1621) amino acids 301-309 ofPRAME ILNSRPPAVLYVDSLFFL (SEQ ID NO: 1622) LYVDSLFFLILNSRPPAVLYVDSLFFL(SEQ ID NO: 1623) amino acids 292-303 of MAGE-6 ILNSRPPAVKISGGPRISYPL(SEQ ID NO: 1624), KISGGPRISYPLILNSRPPAVKISGGPRISYPL (SEQ ID NO: 1625)amino acids 157-167 of NY-ESO-1 ILNSRPPAVSLLMWITQCFL (SEQ ID NO: 1626),SLLMWITQCFLILNSRPPAVVSLLMWITQCFL (SEQ ID NO: 1627) amino acids 157-165of NY-ESO-1 ILNSRPPAVSLLMWITQC (SEQ ID NO: 1628),SLLMWITQCILNSRPPAVSLLMWITQC (SEQ ID NO: 1629) amino acids 155-163 ofNY-ESO-1 ILNSRPPAVQLSLLMWIT (SEQ ID NO: 1630),QLSLLMWITILNSRPPAVQLSLLMWIT (SEQ ID NO: 1631) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVF ILNSRPPAVTSYVKVLHHMVKISG (SEQ ID NO: 1632)amino acids 281-295 of MAGE-3 Analog of I105-V113 of SEQ amino acids161-169 of MAGE-1 IMNSRPPAV EADPTGHSY (SEQ ID NO: 1633), ID NO: 2 havingmethionine EADPTGHSYIMNSRPPAVEADPTGHSY (SEQ ID NO: 1634) substituted forthreonine at the second amino acids residue and alanine substituted forcysteine at the eighth amino acid residue (IMNSRPPAV, SEQ ID NO: 185)amino acids 230-238 of MAGE-1 IMNSRPPAV SAYGEPRKL (SEQ ID NO: 1635),SAYGEPRKLIMNSRPPAV SAYGEPRKL (SEQ ID NO: 1636) amino acids 168-176 ofMAGE-3 IMNSRPPAVEVDPIGHLY (SEQ ID NO: 1637), EVDPIGHLYIMNSRPPAVEVDPIGHLY (SEQ ID NO: 1638) amino acids 271-279 of MAGE-3IMNSRPPAVFLWGPRALV (SEQ ID NO: 1639), FLWGPRALVIMNSRPPAV FLWGPRALV (SEQID NO: 1640) amino acids 167-176 of MAGE-3 IMNSRPPAVMEVDPIGHLY (SEQ IDNO: 1641), MEVDPIGHLYIMNSRPPAVMEVDPIGHLY (SEQ ID NO: 1642) amino acids2-10 of BAGE IMNSRPPAVAARAVFLAL (SEQ ID NO: 1643),AARAVFLALIMNSRPPAVAARAVFLAL (SEQ ID NO: 1644) amino acids 9-16 ofGAGE-1,2 IMNSRPPAVYRPRPRRY (SEQ ID NO: 1645), YRPRPRRYIMNSRPPAVYRPRPRRY(SEQ ID NO: 1646) amino acids 11-20 of RAGE IMNSRPPAVSPSSNRIRNT (SEQ IDNO: 1647), SPSSNRIRNTIMNSRPPAVSPSSNRIRNT (SEQ ID NO: 1648) amino acids23-32 of CDK4 IMNSRPPAVARDPHSGHFV (SEQ ID NO: 1649),ARDPHSGHFVIMNSRPPAVARDPHSGHFV (SEQ ID NO: 1650) amino acids 29-37 ofβ-catenin IMNSRPPAVSYLDSGIHS (SEQ ID NO: 1651),SYLDSGIHSIMNSRPPAVSYLDSGIHS (SEQ ID NO: 1652) amino acids 1-9 ofTyrosinase IMNSRPPAVMLLAVLYCL (SEQ ID NO: 1653),MLLAVLYCLIMNSRPPAVMLLAVLYCL (SEQ ID NO: 1654) amino acids 206-214 ofTyrosinase IMNSRPPAVAFLPWHRLF (SEQ ID NO: 1655),AFLPWHRLFIMNSRPPAVAFLPWHRLF (SEQ ID NO: 1656) amino acids 56-70 ofTyrosinase IMNSRPPAVQNILLSNAPLGPQFP (SEQ ID NO: 1657),QNILLSNAPLGPQFPIMNSRPPAVQNILLSNAPLGPQFP (SEQ ID NO: 1658) amino acids448-462 of Tyrosinase IMNSRPPAVDYSYLQDSDPDSFQD (SEQ ID NO: 1659),DYSYLQDSDPDSFQDIMNSRPPAVDYSYLQDSDPDSFQD (SEQ ID NO: 1660) amino acids32-40 of Melan-A^(MART-1) IMNSRPPAVJLTVILGVL (SEQ ID NO: 1661),JLTVILGVLIMNSRPPAVJLTVILGVL (SEQ ID NO: 1662) amino acids 154-162 ofgp100^(Pme117) IMNSRPPAVKTWGQYWQV (SEQ ID NO: 1663),KTWGQYWQVIMNSRPPAVKTWGQYWQV (SEQ ID NO: 1664) amino acids 209-217 ofgp100^(Pme117) IMNSRPPAVITDQVPFSV (SEQ ID NO: 1665),ITDQVPFSVIMNSRPPAVITDQVPFSV (SEQ ID NO: 1666) amino acids 280-288 ofgp100^(Pme117) IMNSRPPAVYLEPGPVTA (SEQ ID NO: 1667),YLEPGPVTAIMNSRPPAVYLEPGPVTA (SEQ ID NO: 1668) amino acids 457-466 ofgp100^(Pme117) IMNSRPPAVLLDGTATLRL (SEQ ID NO: 1669),LLDGTATLRLIMNSRPPAVLLDGTATLRL (SEQ ID NO: 1670) amino acids 476-485 ofgp100^(Pme117) IMNSRPPAVVLYRYGSFSV (SEQ ID NO: 1671),VLYRYGSFSVIMNSRPPAVVLYRYGSFSV (SEQ ID NO: 1672) amino acids 301-309 ofPRAME IMNSRPPAV LYVDSLFFL (SEQ ID NO: 1673), LYVDSLFFLIMNSRPPAVLYVDSLFFL(SEQ ID NO: 1674) amino acids 292-303 of MAGE-6 IMNSRPPAVKISGGPRISYPL(SEQ ID NO: 1675), KISGGPRISYPLIMNSRPPAVKISGGPRISYPL (SEQ ID NO: 1676)amino acids 157-167 of NY-ESO-1 IMNSRPPAVSLLMWITQCFL (SEQ ID NO: 1677),SLLMWITQCFLITNSRPPAVSLLMWITQCFL (SEQ ID NO: 1678) amino acids 157-165 ofNY-ESO-1 IMNSRPPAVSLLMWITQC (SEQ ID NO: 1679),SLLMWITQCIMNSRPPAVSLLMWITQC (SEQ ID NO: 1680) amino acids 155-163 ofNY-ESO-1 IMNSRPPAVQLSLLMWIT (SEQ ID NO: 1681),QLSLLMWITIMNSRPPAVQLSLLMWIT (SEQ ID NO: 1682) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFIMNSRPPAVTSYVKVLHHMVKISG (SEQ ID NO: 1683)amino acids 281-295 of MAGE-3 Analog of I105-V113 of SEQ amino acids161-169 of MAGE-1 ITNSRPPSV EADPTGHSY (SEQ ID NO: 1684), ID NO: 2 havingserine EADPTGHSYITNSRPPSVEADPTGHSY (SEQ ID NO: 1685) substituted forcysteine at the eighth amino acid residue (ITNSRPPSV, SEQ ID NO: 189)amino acids 230-238 of MAGE-1 ITNSRPPSV SAYGEPRKL (SEQ ID NO: 1686),SAYGEPRKLITNSRPPSV SAYGEPRKL (SEQ ID NO: 1687) amino acids 168-176 ofMAGE-3 ITNSRPPSVEVDPIGHLY (SEQ ID NO: 1688), EVDPIGHLYITNSRPPSVEVDPIGHLY (SEQ ID NO: 1689) amino acids 271-279 of MAGE-3ITNSRPPSVFLWGPRALV (SEQ ID NO: 1690), FLWGPRALVITNSRPPSV FLWGPRALV (SEQID NO: 1691) amino acids 167-176 of MAGE-3 ITNSRPPSVMEVDPIGHLY (SEQ IDNO: 1692), MEVDPIGHLYITNSRPPSVMEVDPIGHLY (SEQ ID NO: 1693) amino acids2-10 of BAGE ITNSRPPSVAARAVFLAL (SEQ ID NO: 1694),AARAVFLALITNSRPPSVAARAVFLAL (SEQ ID NO: 1695) amino acids 9-16 ofGAGE-1,2 ITNSRPPSVYRPRPRRY (SEQ ID NO: 1696), YRPRPRRYITNSRPPSVYRPRPRRY(SEQ ID NO: 1697) amino acids 11-20 of RAGE ITNSRPPSVSPSSNRIRNT (SEQ IDNO: 1698), SPSSNRIRNTITNSRPPSVSPSSNRIRNT (SEQ ID NO: 1699) amino acids23-32 of CDK4 ITNSRPPSVARDPHSGHFV (SEQ ID NO: 1700),ARDPHSGHFVITNSRPPSVARDPHSGHFV (SEQ ID NO: 1701) amino acids 29-37 ofβ-catenin ITNSRPPSVSYLDSGIHS (SEQ ID NO: 1702),SYLDSGIHSITNSRPPSVSYLDSGIHS (SEQ ID NO: 1703) amino acids 1-9 ofTyrosinase ITNSRPPSVMLLAVLYCL (SEQ ID NO: 1704),MLLAVLYCLITNSRPPSVMLLAVLYCL (SEQ ID NO: 1705) amino acids 206-214 ofTyrosinase ITNSRPPSVAFLPWHRLF (SEQ ID NO: 1706),AFLPWHRLFITNSRPPSVAFLPWHRLF (SEQ ID NO: 1707) amino acids 56-70 ofTyrosinase ITNSRPPSVQNILLSNAPLGPQFP (SEQ ID NO: 1708),QNILLSNAPLGPQFPITNSRPPSVQNILLSNAPLGPQFP (SEQ ID NO: 1709) amino acids448-462 of Tyrosinase ITNSRPPSVDYSYLQDSDPDSFQD (SEQ ID NO: 1710),DYSYLQDSDPDSFQDITNSRPPSVDYSYLQDSDPDSFQD (SEQ ID NO: 1711) amino acids32-40 of Melan-A^(MART-1) ITNSRPPSVJLTVILGVL (SEQ ID NO: 1712),JLTVILGVLITNSRPPSVJLTVILGVL (SEQ ID NO: 1713) amino acids 154-162 ofgp100^(Pme117) ITNSRPPSVKTWGQYWQV (SEQ ID NO: 1714),KTWGQYWQVITNSRPPSVKTWGQYWQV (SEQ ID NO: 1715) amino acids 209-217 ofgp100^(Pme117) ITNSRPPSVITDQVPFSV (SEQ ID NO: 1716),ITDQVPFSVITNSRPPSVITDQVPFSV (SEQ ID NO: 1717) amino acids 280-288 ofgp100^(Pme117) ITNSRPPSVYLEPGPVTA (SEQ ID NO: 1718),YLEPGPVTAITNSRPPSVYLEPGPVTA (SEQ ID NO: 1719) amino acids 457-466 ofgp100^(Pme117) ITNSRPPSVLLDGTATLRL (SEQ ID NO: 1720),LLDGTATLRLITNSRPPSVLLDGTATLRL (SEQ ID NO: 1721) amino acids 476-485 ofgp100^(Pme117) ITNSRPPSVVLYRYGSFSV (SEQ ID NO: 1722),VLYRYGSFSVITNSRPPSVVLYRYGSFSV (SEQ ID NO: 1723) amino acids 301-309 ofPRAME ITNSRPPSV LYVDSLFFL (SEQ ID NO: 1724), LYVDSLFFLITNSRPPSVLYVDSLFFL(SEQ ID NO: 1725) amino acids 292-303 of MAGE-6 ITNSRPPSV KISGGPRISYPL(SEQ ID NO: 1726), KISGGPRISYPLITNSRPPSVKISGGPRISYPL (SEQ ID NO: 1727)amino acids 157-167 of NY-ESO-1 ITNSRPPSVSLLMWITQCFL (SEQ ID NO: 1728),SLLMWITQCFLITNSRPPSVSLLMWITQCFL (SEQ ID NO: 1729) amino acids 157-165 ofNY-ESO-1 ITNSRPPSVSLLMWITQC (SEQ ID NO: 1730),SLLMWITQCITNSRPPSVSLLMWITQC (SEQ ID NO: 1731) amino acids 155-163 ofNY-ESO-1 ITNSRPPSVQLSLLMWIT (SEQ ID NO: 1732),QLSLLMWITIMNSRPPSVQLSLLMWIT (SEQ ID NO: 1733) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFITNSRPPSVTSYVKVLHHMVKISG (SEQ ID NO: 1734)amino acids 281-295 of MAGE-3 Analog of K104-V113 of SEQ amino acids161-169 of MAGE-1 KITNSRPPSV EADPTGHSY (SEQ ID NO: 1735), ID NO: 2having serine EADPTGHSYKITNSRPPSVEADPTGHSY (SEQ ID NO: 1736) substitutedfor cysteine at the ninth amino acid residue (KITNSRPPSV, SEQ ID NO:178) amino acids 230-238 of MAGE-1 KITNSRPPSV SAYGEPRKL (SEQ ID NO:1737), SAYGEPRKLKITNSRPPSV SAYGEPRKL (SEQ ID NO: 1738) amino acids168-176 of MAGE-3 KITNSRPPSVEVDPIGHLY (SEQ ID NO: 1739),EVDPIGHLYKITNSRPPSV EVDPIGHLY (SEQ ID NO: 1740) amino acids 271-279 ofMAGE-3 KITNSRPPSVFLWGPRALV (SEQ ID NO: 1741), FLWGPRALVKITNSRPPSVFLWGPRALV (SEQ ID NO: 1742) amino acids 167-176 of MAGE-3KITNSRPPSVMEVDPIGHLY (SEQ ID NO: 1743), MEVDPIGHLYKITNSRPPSVMEVDPIGHLY(SEQ ID NO: 1744) amino acids 2-10 of BAGE KITNSRPPSVAARAVFLAL (SEQ IDNO: 1745), AARAVFLALKITNSRPPSVAARAVFLAL (SEQ ID NO: 1746) amino acids9-16 of GAGE-1,2 KITNSRPPSVYRPRPRRY (SEQ ID NO: 1747),YRPRPRRYKITNSRPPSVYRPRPRRY (SEQ ID NO: 1748) amino acids 11-20 of RAGEKITNSRPPSVSPSSNRIRNT (SEQ ID NO: 1749), SPSSNRIRNTKITNSRPPSVSPSSNRIRNT(SEQ ID NO: 1750) amino acids 23-32 of CDK4 KITNSRPPSVARDPHSGHFV (SEQ IDNO: 1751), ARDPHSGHFVKITNSRPPSVARDPHSGHFV (SEQ ID NO: 1752) amino acids29-37 of β-catenin KITNSRPPSVSYLDSGIHS (SEQ ID NO: 1753),SYLDSGIHSKITNSRPPSVSYLDSGIHS (SEQ ID NO: 1754) amino acids 1-9 ofTyrosinase KITNSRPPSVMLLAVLYCL (SEQ ID NO: 1755),MLLAVLYCLKITNSRPPSVMLLAVLYCL (SEQ ID NO: 1756) amino acids 206-214 ofTyrosinase KITNSRPPSVAFLPWHRLF (SEQ ID NO: 1757),AFLPWHRLFKITNSRPPSVAFLPWHRLF (SEQ ID NO: 1758) amino acids 56-70 ofTyrosinase KITNSRPPSVQNILLSNAPLGPQFP (SEQ ID NO: 1759),QNILLSNAPLGPQFPKITNSRPPSVQNILLSNAPLGPQFP (SEQ ID NO: 1760) amino acids448-462 of Tyrosinase KITNSRPPSVDYSYLQDSDPDSFQD (SEQ ID NO: 1761),DYSYLQDSDPDSFQDKITNSRPPSVDYSYLQDSDPDSFQD (SEQ ID NO: 1762) amino acids32-40 of Melan-A^(MART-1) KITNSRPPSVJLTVILGVL (SEQ ID NO: 1763),JLTVILGVLKITNSRPPSVJLTVILGVL (SEQ ID NO: 1764) amino acids 154-162 ofgp100^(Pme117) KITNSRPPSVKTWGQYWQV (SEQ ID NO: 1765),KTWGQYWQVKITNSRPPSVKTWGQYWQV (SEQ ID NO: 1766) amino acids 209-217 ofgp100^(Pme117) KITNSRPPSVITDQVPFSV (SEQ ID NO: 1767),ITDQVPFSVKITNSRPPSVITDQVPFSV (SEQ ID NO: 1768) amino acids 280-288 ofgp100^(Pme117) KITNSRPPSVYLEPGPVTA (SEQ ID NO: 1769),YLEPGPVTAKITNSRPPSVYLEPGPVTA (SEQ ID NO: 1770) amino acids 457-466 ofgp100^(Pme117) KITNSRPPSVLLDGTATLRL (SEQ ID NO: 1771),LLDGTATLRLKITNSRPPSVLLDGTATLRL (SEQ ID NO: 1772) amino acids 476-485 ofgp100^(Pme117) KITNSRPPSVVLYRYGSFSV (SEQ ID NO: 1773),VLYRYGSFSVKITNSRPPSVVLYRYGSFSV (SEQ ID NO: 1774) amino acids 301-309 ofPRAME KITNSRPPSVLYVDSLFFL (SEQ ID NO: 1775),LYVDSLFFLKITNSRPPSVLYVDSLFFL (SEQ ID NO: 1776) amino acids 292-303 ofMAGE-6 KITNSRPPSVKISGGPRISYPL (SEQ ID NO: 1777),KISGGPRISYPLKITNSRPPSVKISGGPRISYPL (SEQ ID NO: 1778) amino acids 157-167of NY-ESO-1 KITNSRPPSVSLLMWITQCFL (SEQ ID NO: 1779),SLLMWITQCFLKITNSRPPSVSLLMWITQCFL (SEQ ID NO: 1780) amino acids 157-165of NY-ESO-1 KITNSRPPSVSLLMWITQC (SEQ ID NO: 1781),SLLMWITQCKITNSRPPSVSLLMWITQC (SEQ ID NO: 1782) amino acids 155-163 ofNY-ESO-1 KITNSRPPSVQLSLLMWIT (SEQ ID NO: 1783),QLSLLMWITKITNSRPPSVQLSLLMWIT (SEQ ID NO: 1784) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFKITNSRPPSVTSYVKVLHHMVKISG (SEQ ID NO: 1785)amino acids 281-295 of MAGE-3 Analog of G22-C30 of SEQ amino acids161-169 of MAGE-1 GVRIVVEYAEADPTGHSY (SEQ ID NO: 1786), ID NO: 2 havingalanine EADPTGHSYGVRIVVEYAEADPTGHSY (SEQ ID NO: 1787) substituted forcysteine at the ninth amino acid residue (GVRIVVEYA, SEQ ID NO: 161)amino acids 230-238 of MAGE-1 GVRIVVEYASAYGEPRKL (SEQ ID NO: 1788),SAYGEPRKLGVRIVVEYASAYGEPRKL (SEQ ID NO: 1789) amino acids 168-176 ofMAGE-3 GVRIVVEYA EVDPIGHLY (SEQ ID NO: 1790),EVDPIGHLYGVRIVVEYAEVDPIGHLY (SEQ ID NO: 1791) amino acids 271-279 ofMAGE-3 GVRIVVEYAFLWGPRALV (SEQ ID NO: 1792), FLWGPRALVGVRIVVEYAFLWGPRALV(SEQ ID NO: 1793) amino acids 167-176 of MAGE-3 GVRIVVEYAMEVDPIGHLY (SEQID NO: 1794), MEVDPIGHLYGVRIVVEYAMEVDPIGHLY (SEQ ID NO: 1795) aminoacids 2-10 of BAGE GVRIVVEYAAARAVFLAL (SEQ ID NO: 1796),AARAVFLALGVRIVVEYAAARAVFLAL (SEQ ID NO: 1797) amino acids 9-16 ofGAGE-1,2 GVRIVVEYAYRPRPRRY (SEQ ID NO: 1798), YRPRPRRYGVRIVVEYAYRPRPRRY(SEQ ID NO: 1799) amino acids 11-20 of RAGE GVRIVVEYASPSSNRIRNT (SEQ IDNO: 1491), SPSSNRIRNTGVRIVVEYASPSSNRIRNT (SEQ ID NO: 1492) amino acids23-32 of CDK4 GVRIVVEYAARDPHSGHFV (SEQ ID NO: 1493),ARDPHSGHFVGVRIVVEYAARDPHSGHFV (SEQ ID NO: 1494) amino acids 29-37 ofβ-catenin GVRIVVEYASYLDSGIHS (SEQ ID NO: 1495),SYLDSGIHSGVRIVVEYASYLDSGIHS (SEQ ID NO: 1496) amino acids 1-9 ofTyrosinase GVRIVVEYAMLLAVLYCL (SEQ ID NO: 1497),MLLAVLYCLGVRIVVEYAMLLAVLYCL (SEQ ID NO: 1498) amino acids 206-214 ofTyrosinase GVRIVVEYAAFLPWHRLF (SEQ ID NO: 1499),AFLPWHRLFGVRIVVEYAAFLPWHRLF (SEQ ID NO: 2131) amino acids 56-70 ofTyrosinase GVRIVVEYAQNILLSNAPLGPQFP (SEQ ID NO: 2132),QNILLSNAPLGPQFPGVRIVVEYAQNILLSNAPLGPQFP (SEQ ID NO: 2133) amino acids448-462 of Tyrosinase GVRIVVEYADYSYLQDSDPDSFQD (SEQ ID NO: 2134),DYSYLQDSDPDSFQDGVRIVVEYADYSYLQDSDPDSFQD (SEQ ID NO: 2135) amino acids32-40 of Melan-A^(MART-1) GVRIVVEYAJLTVILGVL (SEQ ID NO: 2136),JLTVILGVLGVRIVVEYAJLTVILGVL (SEQ ID NO: 2137) amino acids 154-162 ofgp100^(Pme117) GVRIVVEYAKTWGQYWQV (SEQ ID NO: 2138),KTWGQYWQVGVRIVVEYAKTWGQYWQV (SEQ ID NO: 2139) amino acids 209-217 ofgp100^(Pme117) GVRIVVEYAITDQVPFSV (SEQ ID NO: 2140),ITDQVPFSVGVRIVVEYAITDQVPFSV (SEQ ID NO: 2141) amino acids 280-288 ofgp100^(Pme117) GVRIVVEYAYLEPGPVTA (SEQ ID NO: 1800),YLEPGPVTAGVRIVVEYAYLEPGPVTA (SEQ ID NO: 1801) amino acids 457-466 ofgp100^(Pme117) GVRIVVEYALLDGTATLRL (SEQ ID NO: 1802),LLDGTATLRLGVRIVVEYALLDGTATLRL (SEQ ID NO: 1803) amino acids 476-485 ofgp100^(Pme117) KLENGGFPVVLYRYGSFSV (SEQ ID NO: 1804),VLYRYGSFSVKLENGGFPVVLYRYGSFSV (SEQ ID NO: 1805) amino acids 301-309 ofPRAME GVRIVVEYALYVDSLFFL (SEQ ID NO: 1806), LYVDSLFFLGVRIVVEYALYVDSLFFL(SEQ ID NO: 1807) amino acids 292-303 of MAGE-6 GVRIVVEYAKISGGPRISYPL(SEQ ID NO: 1808), KISGGPRISYPLGVRIVVEYAKISGGPRISYPL (SEQ ID NO: 1809)amino acids 157-167 of NY-ESO-1 GVRIVVEYASLLMWITQCFL (SEQ ID NO: 1810),SLLMWITQCFLGVRIVVEYASLLMWITQCFL (SEQ ID NO: 1811) amino acids 157-165 ofNY-ESO-1 GVRIVVEYASLLMWITQC (SEQ ID NO: 1812), SLLMWITQCGVRIVVEYASLLMWITQC (SEQ ID NO: 1813) amino acids 155-163 of NY-ESO-1GVRIVVEYAQLSLLMWIT (SEQ ID NO: 1814), QLSLLMWIT GVRIVVEYAQLSLLMWIT (SEQID NO: 1815) amino acids 157-170 of NY-ESO-1and SLLMWITQCFLPVFGVRIVVEYATSYVKVLHHMVKISG (SEQ ID NO: 1816) amino acids 281-295 of MAGE-3Analog of I25-C33 of SEQ ID amino acids 161-169 of MAGE-1IVVEYAEPAEADPTGHSY (SEQ ID NO: 1817), NO: 2 having alanineEADPTGHSYIVVEYAEPAEADPTGHSY (SEQ ID NO: 1818) substituted for cysteineat the sixth and ninth amino acids residues (IVVEYAEPA, SEQ ID NO: 167)amino acids 230-238 of MAGE-1 IVVEYAEPASAYGEPRKL (SEQ ID NO: 1819),SAYGEPRKLIVVEYAEPASAYGEPRKL (SEQ ID NO: 1820) amino acids 168-176 ofMAGE-3 IVVEYAEPAEVDPIGHLY (SEQ ID NO: 1821), EVDPIGHLYIVVEYAEPAEVDPIGHLY(SEQ ID NO: 1822) amino acids 271-279 of MAGE-3 IVVEYAEPAFLWGPRALV (SEQID NO: 1823), FLWGPRALVIVVEYAEPAFLWGPRALV (SEQ ID NO: 1824) amino acids167-176 of MAGE-3 IVVEYAEPAMEVDPIGHLY (SEQ ID NO: 1825),MEVDPIGHLYIVVEYAEPAMEVDPIGHLY (SEQ ID NO: 1826) amino acids 2-10 of BAGEIVVEYAEPAAARAVFLAL (SEQ ID NO: 1827), AARAVFLALIVVEYAEPAAARAVFLAL (SEQID NO: 1828) amino acids 9-16 of GAGE-1,2 IVVEYAEPAYRPRPRRY (SEQ ID NO:1829), YRPRPRRYIVVEYAEPAYRPRPRRY (SEQ ID NO: 1830) amino acids 11-20 ofRAGE IVVEYAEPASPSSNRIRNT (SEQ ID NO: 1831),SPSSNRIRNTIVVEYAEPASPSSNRIRNT (SEQ ID NO: 1832) amino acids 23-32 ofCDK4 IVVEYAEPAARDPHSGHFV (SEQ ID NO: 1833),ARDPHSGHFVIVVEYAEPAARDPHSGHFV (SEQ ID NO: 1834) amino acids 29-37 ofβ-catenin IVVEYAEPASYLDSGIHS (SEQ ID NO: 1835),SYLDSGIHSIVVEYAEPASYLDSGIHS (SEQ ID NO: 1836) amino acids 1-9 ofTyrosinase IVVEYAEPAMLLAVLYCL (SEQ ID NO: 1837),MLLAVLYCLIVVEYAEPAMLLAVLYCL (SEQ ID NO: 1838) amino acids 206-214 ofTyrosinase IVVEYAEPAAFLPWHRLF (SEQ ID NO: 1839),AFLPWHRLFIVVEYAEPAAFLPWHRLF (SEQ ID NO: 1840) amino acids 56-70 ofTyrosinase IVVEYAEPAQNILLSNAPLGPQFP (SEQ ID NO: 1841),QNILLSNAPLGPQFPIVVEYAEPAQNILLSNAPLGPQFP (SEQ ID NO: 1842) amino acids448-462 of Tyrosinase IVVEYAEPADYSYLQDSDPDSFQD (SEQ ID NO: 1843),DYSYLQDSDPDSFQDIVVEYAEPADYSYLQDSDPDSFQD (SEQ ID NO: 1844) amino acids32-40 of Melan-A^(MART-1) IVVEYAEPAVJLTVILGVL (SEQ ID NO: 1845),JLTVILGVLIVVEYAEPAJLTVILGVL (SEQ ID NO: 1846) amino acids 154-162 ofgp100^(Pme117) IVVEYAEPAKTWGQYWQV (SEQ ID NO: 1847),KTWGQYWQVIVVEYAEPAKTWGQYWQV (SEQ ID NO: 1848) amino acids 209-217 ofgp100^(Pme117) IVVEYAEPAITDQVPFSV (SEQ ID NO: 1849),ITDQVPFSVIVVEYAEPAITDQVPFSV (SEQ ID NO: 1850) amino acids 280-288 ofgp100^(Pme117) IVVEYAEPAYLEPGPVTA (SEQ ID NO: 1851),YLEPGPVTAIVVEYAEPAYLEPGPVTA (SEQ ID NO: 1852) amino acids 457-466 ofgp100^(Pme117) IVVEYAEPALLDGTATLRL (SEQ ID NO: 1853),LLDGTATLRLIVVEYAEPALLDGTATLRL (SEQ ID NO: 1854) amino acids 476-485 ofgp100^(Pme117) IVVEYAEPAVLYRYGSFSV (SEQ ID NO: 1855),VLYRYGSFSVIVVEYAEPAVLYRYGSFSV (SEQ ID NO: 1856) amino acids 301-309 ofPRAME IVVEYAEPALYVDSLFFL (SEQ ID NO: 1857), LYVDSLFFLIVVEYAEPALYVDSLFFL(SEQ ID NO: 1858) amino acids 292-303 of MAGE-6 IVVEYAEPAKISGGPRISYPL(SEQ ID NO: 1859), KISGGPRISYPLIVVEYAEPAKISGGPRISYPL (SEQ ID NO: 1860)amino acids 157-167 of NY-ESO-1 IVVEYAEPASLLMWITQCFL (SEQ ID NO: 1861),SLLMWITQCFLIVVEYAEPASLLMWITQCFL (SEQ ID NO: 1862) amino acids 157-165 ofNY-ESO-1 IVVEYAEPASLLMWITQC (SEQ ID NO: 1863),SLLMWITQCIVVEYAEPASLLMWITQC (SEQ ID NO: 1864) amino acids 155-163 ofNY-ESO-1 IVVEYAEPAQLSLLMWIT (SEQ ID NO: 1865), QLSLLMWITIVVEYAEPAQLSLLMWIT (SEQ ID NO: 1866) amino acids 157-170 of NY-ESO-1 andSLLMWITQCFLPVF IVVEYAEPATSYVKVLHHMVKISG (SEQ ID NO: 1867) amino acids281-295 of MAGE-3 Analog of K104-C112 of SEQ amino acids 161-169 ofMAGE-1 KITNSRPPAEADPTGHSY (SEQ ID NO: 1868), ID NO: 2 having alanineEADPTGHSY KITNSRPPAEADPTGHSY (SEQ ID NO: 1869) substituted for cysteineat the ninth amino acid residue (KITNSRPPA, SEQ ID NO: 173) amino acids230-238 of MAGE-1 KITNSRPPASAYGEPRKL (SEQ ID NO: 1870),SAYGEPRKLKITNSRPPASAYGEPRKL (SEQ ID NO: 1871) amino acids 168-176 ofMAGE-3 KITNSRPPAEVDPIGHLY (SEQ ID NO: 1872), EVDPIGHLYKITNSRPPAEVDPIGHLY(SEQ ID NO: 1873) amino acids 271-279 of MAGE-3 KITNSRPPAFLWGPRALV (SEQID NO: 1874), FLWGPRALVKITNSRPPAFLWGPRALV (SEQ ID NO: 1875) amino acids167-176 of MAGE-3 KITNSRPPAMEVDPIGHLY (SEQ ID NO: 1876),MEVDPIGHLYKITNSRPPAMEVDPIGHLY (SEQ ID NO: 1877) amino acids 2-10 of BAGEKITNSRPPAAARAVFLAL (SEQ ID NO: 1878), AARAVFLALKITNSRPPAAARAVFLAL (SEQID NO: 1879) amino acids 9-16 of GAGE-1,2 KITNSRPPAYRPRPRRY (SEQ ID NO:1880), YRPRPRRYKITNSRPPAYRPRPRRY (SEQ ID NO: 1881) amino acids 11-20 ofRAGE KITNSRPPASPSSNRIRNT (SEQ ID NO: 1882),SPSSNRIRNTKITNSRPPASPSSNRIRNT (SEQ ID NO: 1883) amino acids 23-32 ofCDK4 KITNSRPPAARDPHSGHFV (SEQ ID NO: 1884),ARDPHSGHFVKITNSRPPAARDPHSGHFV (SEQ ID NO: 1885) amino acids 29-37 ofβ-catenin KITNSRPPASYLDSGIHS (SEQ ID NO: 1886),SYLDSGIHSKITNSRPPASYLDSGIHS (SEQ ID NO: 1887) amino acids 1-9 ofTyrosinase KITNSRPPAMLLAVLYCL (SEQ ID NO: 1888),MLLAVLYCLKITNSRPPAMLLAVLYCL (SEQ ID NO: 1889) amino acids 206-214 ofTyrosinase KITNSRPPAAFLPWHRLF (SEQ ID NO: 1890),AFLPWHRLFKITNSRPPAAFLPWHRLF (SEQ ID NO: 1891) amino acids 56-70 ofTyrosinase KITNSRPPAQNILLSNAPLGPQFP (SEQ ID NO: 1892), QNILLSNAPLGPQFPKITNSRPPAQNILLSNAPLGPQFP (SEQ ID NO: 1893) amino acids 448-462 ofTyrosinase KITNSRPPADYSYLQDSDPDSFQD (SEQ ID NO: 1894),DYSYLQDSDPDSFQDKITNSRPPADYSYLQDSDPDSFQD (SEQ ID NO: 1895) amino acids32-40 of Melan-A^(MART-1) KITNSRPPAJLTVILGVL (SEQ ID NO: 1896),JLTVILGVLKITNSRPPAJLTVILGVL (SEQ ID NO: 1897) amino acids 154-162 ofgp100^(Pme117) KITNSRPPAKTWGQYWQV (SEQ ID NO: 1898),KTWGQYWQVKITNSRPPAKTWGQYWQV (SEQ ID NO: 1899) amino acids 209-217 ofgp100^(Pme117) KITNSRPPAITDQVPFSV (SEQ ID NO: 1900),ITDQVPFSVKITNSRPPAITDQVPFSV (SEQ ID NO: 1901) amino acids 280-288 ofgp100^(Pme117) KITNSRPPAYLEPGPVTA (SEQ ID NO: 1902),YLEPGPVTAKITNSRPPAYLEPGPVTA (SEQ ID NO: 1903) amino acids 457-466 ofgp100^(Pme117) KITNSRPPALLDGTATLRL (SEQ ID NO: 1904),LLDGTATLRLKITNSRPPALLDGTATLRL (SEQ ID NO: 1905) amino acids 476-485 ofgp100^(Pme117) KITNSRPPAVLYRYGSFSV (SEQ ID NO: 1906),VLYRYGSFSVKITNSRPPAVLYRYGSFSV (SEQ ID NO: 1907) amino acids 301-309 ofPRAME KITNSRPPALYVDSLFFL (SEQ ID NO: 1908), LYVDSLFFLKITNSRPPALYVDSLFFL(SEQ ID NO: 1909) amino acids 292-303 of MAGE-6 KITNSRPPAKISGGPRISYPL(SEQ ID NO: 1910), KISGGPRISYPLKITNSRPPAKISGGPRISYPL (SEQ ID NO: 1911)amino acids 157-167 of NY-ESO-1 KITNSRPPASLLMWITQCFL (SEQ ID NO: 1912),SLLMWITQCFLKITNSRPPASLLMWITQCFL (SEQ ID NO: 1913) amino acids 157-165 ofNY-ESO-1 KITNSRPPASLLMWITQC (SEQ ID NO: 1914),SLLMWITQCKITNSRPPASLLMWITQC (SEQ ID NO: 1915) amino acids 155-163 ofNY-ESO-1 KITNSRPPAQLSLLMWIT (SEQ ID NO: 1916),QLSLLMWITKITNSRPPAQLSLLMWIT (SEQ ID NO: 1917) amino acids 157-170 ofNY-ESO-1 and SLLMWITQCFLPVFKITNSRPPATSYVKVLHHMVKISG (SEQ ID NO: 1918)amino acids 281-295 of MAGE-3

TABLE D Exemplary Cell-Penetrating C35 Epitope Peptide Sequence (CPP)Exemplary Polypeptide Containing C35 Epitope and CPP S9-V17 of SEQ IDNO: 2 HSV-1 tegument protein VP22SVAPPPEEVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRSVAPPPEEVFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1919),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVESVAPPPEEVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1920)membrane-translocating sequence SVAPPPEEVAAVLLPVLLAAP (SEQ ID NO: 1921),(MST) from h region of the signal AAVLLPVLLAAPSVAPPPEEVAAVLLPVLLAAP (SEQID NO: 1922) sequence of Kaposi fibroblast growth factor S21-Y29 of SEQID NO: 2 HSV-1 tegument protein VP22SGVRIVVEYMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRSGVRIVVEYFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1923),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVESGVRIVVEYMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1924)membrane-translocating sequence SGVRIVVEYAAVLLPVLLAAP (SEQ ID NO: 1925),(MST) from h region of the signal AAVLLPVLLAAPSGVRIVVEYAAVLLPVLLAAP (SEQID NO: 1926) sequence of Kaposi fibroblast growth factor G22-C30 of SEQID NO: 2 HSV-1 tegument protein VP22GVRIVVEYCMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVGVRIVVEYCRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:1927), MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEGVRIVVEYCMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1928)membrane-translocating sequence GVRIVVEYCAAVLLPVLLAAP (SEQ ID NO: 1929),(MST) from h region of the signal AAVLLPVLLAAPGVRIVVEYCAAVLLPVLLAAP (SEQID NO: 1930) sequence of Kaposi fibroblast growth factor I25-C33 of SEQID NO: 2 HSV-1 tegument protein VP22IVVEYCEPCMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRIVVEYCEPCFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1931),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEIVVEYCEPCMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1932)membrane-translocating sequence IVVEYCEPCAAVLLPVLLAAP (SEQ ID NO: 1933),(MST) from h region of the signal AAVLLPVLLAAPIVVEYCEPCAAVLLPVLLAAP (SEQID NO: 1934) sequence of Kaposi fibroblast growth factor T38-V46 of SEQID NO: 2 HSV-1 tegument protein VP22TYLELASAVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVTYLELASAVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:1935), MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVETYLELASAVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1936)membrane-translocating sequence TYLELASAVAAVLLPVLLAAP (SEQ ID NO: 1937),(MST) from h region of the signal AAVLLPVLLAAP TYLELASAVAAVLLPVLLAAP(SEQ ID NO: 1938) sequence of Kaposi fibroblast growth factor G61-I69 ofSEQ ID NO: 2 HSV-1 tegument protein VP22GTGAFEIEIMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRGTGAFEIEIFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1939),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEGTGAFEIEIMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1940)membrane-translocating sequence GTGAFEIEIAAVLLPVLLAAP (SEQ ID NO: 1941),(MST) from h region of the signal AAVLLPVLLAAPGTGAFEIEIAAVLLPVLLAAP (SEQID NO: 1942) sequence of Kaposi fibroblast growth factor F65-L73 of SEQID NO: 2 HSV-1 tegument protein VP22FEIEINGQLMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFEIEINGQLFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1943),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEFEIEINGQLMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1944)membrane-translocating sequence FEIEINGQLAAVLLPVLLAAP (SEQ ID NO: 1945),(MST) from h region of the signal AAVLLPVLLAAPFEIEINGQLAAVLLPVLLAAP (SEQID NO: 1946) sequence of Kaposi fibroblast growth factor I67-F75 of SEQID NO: 2 HSV-1 tegument protein VP22IEINGQLVFMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRIEINGQLVFFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1947),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEIEINGQLVFMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1948)membrane-translocating sequence IEINGQLVFAAVLLPVLLAAP (SEQ ID NO: 1949),(MST) from h region of the signal AAVLLPVLLAAPIEINGQLVFAAVLLPVLLAAP (SEQID NO: 1950) sequence of Kaposi fibroblast growth factor K77-Y85 of SEQID NO: 2 HSV-1 tegument protein VP22KLENGGFPYMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVKLENGGFPYRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:1951), MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEKLENGGFPYMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1952)membrane-translocating sequence KLENGGFPYAAVLLPVLLAAP (SEQ ID NO: 1953),(MST) from h region of the signal AAVLLPVLLAAPKLENGGFPYAAVLLPVLLAAP (SEQID NO: 1954) sequence of Kaposi fibroblast growth factor Q72-E86 of SEQID NO: 2 HSV-1 tegument protein VP22QLVFSKLENGGFPYEMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSQLVFSKLENGGFPYERQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:1955), MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRIPVEQLVFSKLENGGFPYEMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1956)membrane-translocating sequence QLVFSKLENGGFPYEAAVLLPVLLAAP (SEQ ID NO:1957), (MST) from h region of the signalAAVLLPVLLAAPQLVFSKLENGGFPYEAAVLLPVLLAAP (SEQ ID NO: 1958) sequence ofKaposi fibroblast growth factor G81-L89 of SEQ ID NO: 2 HSV-1 tegumentprotein VP22GGFPYEKDLMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVGGFPYEKDLRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:1959), MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRLPVEGGFPYEKDLMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1960)membrane-translocating sequence GGFPYEKDLAAVLLPVLLAAP (SEQ ID NO: 1961),(MST) from h region of the signal AAVLLPVLLAAPGGFPYEKDLAAVLLPVLLAAP (SEQID NO: 1962) sequence of Kaposi fibroblast growth factor K104-C112 ofSEQ ID NO: 2 HSV-1 tegument protein VP22KITNSRPPCMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRKITNSRPPCFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1963),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEKITNSRPPCMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1964)membrane-translocating sequence KITNSRPPCAAVLLPVLLAAP (SEQ ID NO: 1965),(MST) from h region of the signal AAVLLPVLLAAPKITNSRPPCAAVLLPVLLAAP (SEQID NO: 1966) sequence of Kaposi fibroblast growth factor K104-V113 ofSEQ ID NO: 2 HSV-1 tegument protein VP22KITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVKITNSRPPCVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:1967), MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRPVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1968)membrane-translocating sequence KITNSRPPCVAAVLLPVLLAAP (SEQ ID NO:1969), (MST) from h region of the signalAAVLLPVLLAAPKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO: 1970) sequence of Kaposifibroblast growth factor I105-V113 of SEQ ID NO: 2 HSV-1 tegumentprotein VP22ITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRITNSRPPCV FVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1971),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1972)membrane-translocating sequence ITNSRPPCVAAVLLPVLLAAP (SEQ ID NO: 1973),(MST) from h region of the signal AAVLLPVLLAAPITNSRPPCVAAVLLPVLLAAP (SEQID NO: 1974) sequence of Kaposi fibroblast growth factor T101-V113 ofSEQ ID NO: 2 HSV-1 tegument protein VP22TLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRLEKITNSRPPCVILGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:1975), MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1976)membrane-translocating sequence TLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO:1977), (MST) from h region of the signalAAVLLPVLLAAPTLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO: 1978) sequence ofKaposi fibroblast growth factor I93-V113 of SEQ ID NO: 2 HSV-1 tegumentprotein VP22IRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGAIRRASNGETLEKITNSRPPCVLQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQID NO: 1979),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1980)membrane-translocating sequence IRRASNGETLEKITNSRPPCVAAVLLPVLLAAP (SEQID NO: 1981), (MST) from h region of the signalAAVLLPVLLAAPIRRASNGETLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO: 1982) sequenceof Kaposi fibroblast growth factor D88-V113 of SEQ ID NO: 2 HSV-1tegument protein VP22DLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTDLIEAIRRASNGETLEKITNSSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPP RPPCVPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRP RRPVE(SEQ ID NO: 1983),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1984)membrane-translocating sequence DLIEAIRRASNGETLEKITNSRPPCVAAVLLPVLLAAP(SEQ ID NO: 1985), (MST) from h region of the signalAAVLLPVLLAAPDLIEAIRRASNGETLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO: 1986)sequence of Kaposi fibroblast growth factor P84-V113 of SEQ ID NO: 2HSV-1 tegument protein VP22PYEKDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPYEKDLIEAIRRASNGETLEPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARKITNSRPPCVAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1987),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEPYEKDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1988)membrane-translocating sequencePYEKDLIEAIRRASNGETLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO: 1989), (MST) fromh region of the signalAAVLLPVLLAAPPYEKDLIEAIRRASNGETLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO: 1990)sequence of Kaposi fibroblast growth factor K77-V113 of SEQ ID NO: 2HSV-1 tegument protein VP22KLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSKLENGGFPYEKDLIEAIRRASGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVSNGETLEKITNSRPPCVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1991),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:1992) membrane-translocating sequenceKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO: 1993),(MST) from h region of the signalAAVLLPVLLAAPKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV AAVLLPVLLAAP (SEQ IDNO: 1994) sequence of Kaposi fibroblast growth factor Q72-V113 of SEQ IDNO: 2 HSV-1 tegument protein VP22QLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLQLVFSKLENGGFPYEKDLIEYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPAIRRASNGETLEKITNSRPPCVVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1995),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQID NO: 1996) membrane-translocating sequenceQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO:1997), (MST) from h region of the signalAAVLLPVLLAAPQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV sequence ofKaposi fibroblast AAVLLPVLLAAP (SEQ ID NO: 1998) growth factor F65-V113of SEQ ID NO: 2 HSV-1 tegument protein VP22FEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVFEIEINGQLVFSKLENGGFPPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPYEKDLIEAIRRASNGETLEKIEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKTNSRPPCVSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 1999),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2000) membrane-translocating sequenceFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAAVLLPVLLAAP (SEQ IDNO: 2001), (MST) from h region of the signalAAVLLPVLLAAPFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCV sequenceof Kaposi fibroblast AAVLLPVLLAAP (SEQ ID NO: 2002) growth factorL59-V113 of SEQ ID NO: 2 HSV-1 tegument protein VP22LGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSLGGTGAFEIEINGQLVFSKLGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEENGGFPYEKDLIEAIRRASNDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETGETLEKITNSRPPCVILTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2003),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVELGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2004) membrane-translocating sequenceLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRPPCVAAVLLPVLL (MST)from h region of the signal AAP (SEQ ID NO: 2005), sequence of KaposifibroblastAVLLPVLLAAPLGGTGAFEIEINGQLVFSKLENGGFPYEKDLIEAIRRASNGETLEKITNSRP growthfactor PCVAAVLLPVLLAAP (SEQ ID NO: 2006) G99-V113 of SEQ ID NO: 2 HSV-1tegument protein VP22GETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRGETLEKITNSRPPCVQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:2007), MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEGETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2008)membrane-translocating sequence GETLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO:2009), (MST) from h region of the signalAAVLLPVLLAAPGETLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO: 2010) sequence ofKaposi fibroblast growth factor E100-V113 of SEQ ID NO: 2 HSV-1 tegumentprotein VP22ETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQETLEKITNSRPPCVRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:2011), MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEETLEKITNSRPPCVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2012)membrane-translocating sequence ETLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO:2013), (MST) from h region of the signalAAVLLPVLLAAPETLEKITNSRPPCVAAVLLPVLLAAP (SEQ ID NO: 2014) sequence ofKaposi fibroblast growth factor

TABLE E Exemplary Cell-Penetrating C35 Epitope AnalogPeptide Sequence (CPP)Exemplary Polypeptide Containing C35 Epitope Analog and CPPAnalog of K77-Y85 of SEQ ID HSV-1 tegument protein VP22KLENGGFPVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVNO: 2 having valine substitutedRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRfor tyrosine at ninth amino acidAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPresidue (KLENGGFPV, SEQ IDDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCE NO: 171)GKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2015),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEKLENGGFPVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2016)membrane-translocating sequence KLENGGFPVAAVLLPVLLAAP (SEQ ID NO: 2017),(MST) from h region of the signalAAVLLPVLLAAPKLENGGFPVAAVLLPVLLAAP (SEQ ID NO: 2018)sequence of Kaposi fibroblast growth factor Analog of K104-C112 of SEQHSV-1 tegument protein VP22KITNSRPPLMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRID NO: 2 having leucineFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAsubstituted for cysteine at thePRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDninth amino acid residueAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEG(KITNSRPPL, SEQ ID NO:KNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2019),172) MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEKITNSRPPLMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2020)membrane-translocating sequence KITNSRPPLAAVLLPVLLAAP (SEQ ID NO: 2021),(MST) from h region of the signalAAVLLPVLLAAPKITNSRPPLAAVLLPVLLAAP (SEQ ID NO: 2022)sequence of Kaposi fibroblast growth factor Aanalog of I105-V113 of SEQHSV-1 tegument protein VP22ILNSRPPAVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRID NO: 2 having leucineFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAsubstituted for threonine at thePRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDsecond amino acid residue andAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGalanine substituted for cysteineKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2023),at the eighth amino acid residueMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDY(ILNSRPPCV, SEQ ID NO:ALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATK 179)APAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEILNSRPPAVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2024)membrane-translocating sequence ILNSRPPAVAAVLLPVLLAAP (SEQ ID NO: 2025),MST) from h region of the signalAAVLLPVLLAAPILNSRPPAVAAVLLPVLLAAP (SEQ ID NO: 2026)sequence of Kaposi fibroblast growth factor Analog of I105-V113 of SEQHSV-1 tegument protein VP22IMNSRPPAVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRID NO: 2 having methionineFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAsubstituted for threonine at thePRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDsecond amino acid residue andAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGalanine substituted for cysteineKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2027),at the eighth amino acid residueMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDY(IMNSRPPCV, SEQ ID NO:ALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATK 180)APAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEIMNSRPPAVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2028)membrane-translocating sequence IMNSRPPAVAAVLLPVLLAAP (SEQ ID NO: 2029),(MST) from h region of the signalAAVLLPVLLAAPIMNSRPPAVAAVLLPVLLAAP (SEQ ID NO: 2030)sequence of Kaposi fibroblast growth factor Analog of I105-V113 of SEQHSV-1 tegument protein VP22 ITNSRPPSV ID NO: 2 having serineMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYsubstituted for cysteine at theALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKeighth amino acid residueAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAG(ITNSRPPSV, SEQ ID NO:FNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANEL 189)VNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2031),MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEITNSRPPSVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2032)membrane-translocating sequence ITNSRPPSVAAVLLPVLLAAP (SEQ ID NO: 2033),(MST) from h region of the signalAAVLLPVLLAAPITNSRPPSVAAVLLPVLLAAP (SEQ ID NO: 2034)sequence of Kaposi fibroblast growth factor Analog of K104-V113 of SEQHSV-1 tegument protein VP22KITNSRPPSVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVID NO: 2 having serineRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRsubstituted for cysteine at theAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPninth amino acid residueDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCE(KITNSRPPSV, SEQ ID NO:GKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2035),178) MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEKITNSRPPSVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2036)membrane-translocating sequenceKITNSRPPSVAAVLLPVLLAAP (SEQ ID NO: 2037),(MST) from h region of the signalAAVLLPVLLAAPKITNSRPPSVAAVLLPVLLAAP (SEQ ID NO: 2038)sequence of Kaposi fibroblast growth factor Analog of G22 to C30 of SEQHSV-1 tegument protein VP22GVRIVVEYAMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVID NO: 2 having alanineRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRsubstituted for cysteine at ninthAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPamino acid residueDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCE(GVRIVVEYA, SEQ ID NO:GKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2039),161) MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEGVRIVVEYAMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2040)membrane-translocating sequence GVRIVVEYAAAVLLPVLLAAP (SEQ ID NO: 2041),(MST) from h region of the signalAAVLLPVLLAAPGVRIVVEYAAAVLLPVLLAAP (SEQ ID NO: 2042)sequence of Kaposi fibroblast growth factorAnalog of I25 to C33 of SEQ ID HSV-1 tegument protein VP22IVVEYAEPAMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRNO: 2 having alanine substitutedFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAfor cysteine at the sixth andPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDninth amino acid residuesAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEG(IVVEYAEPA, SEQ ID NO:KNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2043),167) MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEIVVEYAEPAMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2044)membrane-translocating sequence IVVEYAEPAAAVLLPVLLAAP (SEQ ID NO: 2045),(MST) from h region of the signalAAVLLPVLLAAPIVVEYAEPAAAVLLPVLLAAP (SEQ ID NO: 2046)sequence of Kaposi fibroblast growth factor Analog of K104-C112 of SEQHSV-1 tegument protein VP22KITNSRPPAMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRID NO: 2 having alanineFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAsubstituted for cysteine at thePRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDninth amino acid residueAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEG(KITNSRPPA, SEQ ID NO:KNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2047),173) MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHRMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEKITNSRPPAMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2048)membrane-translocating sequence KITNSRPPAAAVLLPVLLAAP (SEQ ID NO: 2049),(MST) from h region of the signalAAVLLPVLLAAP KITNSRPPAAAVLLPVLLAAP (SEQ ID NO: 2050)sequence of Kaposi fibroblast growth factor Analog of K104-V113 of SEQHSV-1 tegument protein VP22KITNSRPPAVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVID NO: 2 having alanineRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRsubstituted for cysteine at theAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPninth amino acid residueDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCE(KITNSRPPAV, SEQ ID NO:GKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2051),176) MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVEKITNSRPPAVMTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 2052)membrane-translocating sequenceKITNSRPPAVAAVLLPVLLAAP (SEQ ID NO: 2053),(MST) from h region of the signalAAVLLPVLLAAP KITNSRPPAVAAVLLPVLLAAP (SEQ ID NO: 2054)sequence of Kaposi fibroblast growth factor

The peptides in accordance with the invention can be a variety oflengths, and either in their neutral (uncharged) forms or in forms whichare salts. The peptides in accordance with the invention can containmodifications such as glycosylation, side chain oxidation, orphosphorylation, generally subject to the condition that modificationsdo not destroy the biological activity of the peptides.

The peptides of the invention can be prepared in a wide variety of ways.For the preferred relatively short size, the peptides can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. (See, for example,Stewart & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce ChemicalCo., 1984). Further, individual C35 peptide epitopes and C35 peptideepitope analogs can be joined using chemical ligation to produce largerhomopolymer or heteropolymer polypeptides that are still within thebounds of the invention.

Alternatively, recombinant DNA technology can be employed wherein anucleotide sequence which encodes an immunogenic peptide of interest isinserted into an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression. These procedures are generally known in the art, asdescribed generally in Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus,recombinant polypeptides, which comprise one or more peptide epitopesequences of the invention, can be used to present the appropriate Tcell epitope.

The nucleotide coding sequence for C35 peptide epitopes or C35 peptideepitope analogs of the preferred lengths contemplated herein can besynthesized by chemical techniques, for example, the phosphotriestermethod of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981). Peptideanalogs can be made simply by substituting the appropriate and desirednucleic acid base(s) for those that encode the native peptide sequence;exemplary nucleic acid substitutions are those that encode an amino aciddefined by the motifs/supermotifs herein. The coding sequence can thenbe provided with appropriate linkers and ligated into expression vectorscommonly available in the art, and the vectors used to transformsuitable hosts to produce the desired fusion protein. A number of suchvectors and suitable host systems are now available. For expression ofthe fusion proteins, the coding sequence will be provided with operablylinked start and stop codons, promoter and terminator regions andusually a replication system to provide an expression vector forexpression in the desired cellular host. For example, promoter sequencescompatible with bacterial hosts are provided in plasmids containingconvenient restriction sites for insertion of the desired codingsequence. The resulting expression vectors are transformed into suitablebacterial hosts. Of course, yeast, insect or mammalian cell hosts mayalso be used, employing suitable vectors and control sequences.

It is generally preferable that the peptide epitope be as small aspossible while still maintaining substantially all of the immunologicactivity of the native protein. When possible, it may be desirable tooptimize HLA class I binding peptide epitopes of the invention to alength of about 8 to about 13 amino acid residues, preferably 9 to 10.It is to be appreciated that a longer polypeptide, e.g., a C35polypeptide fragment or a synthetic polypeptide, can comprise one ormore C35 peptide epitopes or C35 peptide epitope analogs in this sizerange (see the Definition Section for the term “epitope” for furtherdiscussion of peptide length). HLA class II binding epitopes arepreferably optimized to a length of about 6 to about 30 amino acids inlength, preferably to between about 13 and about 20 residues.Preferably, the epitopes are commensurate in size with endogenouslyprocessed pathogen-derived peptides or tumor cell peptides that arebound to the relevant HLA molecules. The identification and preparationof peptides of various lengths can be carried out using the techniquesdescribed herein.

An alternative preferred embodiment of the invention comprisesadministration of peptides of the invention linked as a polyepitopicpolypeptide, e.g., homopolymers or heteropolymers, or as a minigene thatencodes a polyepitopic polypeptide.

Another preferred embodiment is obtained by identifying native C35polypeptide regions that contain a high concentration of class I and/orclass II C35 peptide epitopes. Such a sequence is generally selected onthe basis that it contains the greatest number of C35 epitopes per aminoacid length. It is to be appreciated that epitopes can be present in aframe-shifted manner, e.g. a 10 amino acid long peptide could containtwo 9 amino acid long epitopes and one 10 amino acid long epitope; uponintracellular processing, each epitope can be exposed and bound by anHLA molecule upon administration of such a peptide. Thus a larger,preferably multi-epitopic, polypeptide can be generated synthetically,recombinantly, or via cleavage from the native source.

Assays to Detect T-Cell Responses

Once HLA binding peptides are identified, they can be tested for theability to elicit a T-cell response. The preparation and evaluation ofmotif-bearing peptides are described, e.g., in PCT publications WO94/20127 and WO 94/03205, the entire contents of which are herebyincorporated by reference. Briefly, peptides comprising epitopes from aparticular antigen are synthesized and tested for their ability to bindto relevant HLA proteins. These assays may involve evaluation of peptidebinding to purified HLA class I molecules in relation to the binding ofa radioiodinated reference peptide. Alternatively, cells expressingempty class I molecules (i.e. cell surface HLA molecules that lack anybound peptide) may be evaluated for peptide binding by immunofluorescentstaining and flow microfluorimetry. Other assays that may be used toevaluate peptide binding include peptide-dependent class I assemblyassays and/or the inhibition of CTL recognition by peptide competition.Those peptides that bind to an HLA class I molecule, typically with anaffinity of 500 nM or less, are further evaluated for their ability toserve as targets for CTLs derived from infected or immunizedindividuals, as well as for their capacity to induce primary in vitro orin vivo CTL responses that can give rise to CTL populations capable ofreacting with selected target cells associated with pathology.

Analogous assays are used for evaluation of HLA class II bindingpeptides. HLA class II motif-bearing peptides that are shown to bind,typically at an affinity of 1000 nM or less, are further evaluated forthe ability to stimulate HTL responses.

Conventional assays utilized to detect T cell responses includeproliferation assays, lymphokine secretion assays, direct cytotoxicityassays, and limiting dilution assays. For example, antigen-presentingcells that have been incubated with a peptide can be assayed for theability to induce CTL responses in responder cell populations.Antigen-presenting cells can be normal cells such as peripheral bloodmononuclear cells or dendritic cells. Alternatively, mutant, non-humanmammalian cell lines that have been transfected with a human class I MHCgene, and that are deficient in their ability to load class I moleculeswith internally processed peptides, are used to evaluate the capacity ofthe peptide to induce in vitro primary CTL responses. Peripheral bloodmononuclear cells (PBMCs) can be used as the source of CTL precursors.Antigen presenting cells are incubated with peptide, after which thepeptide-loaded antigen-presenting cells are then incubated with theresponder cell population under optimized culture conditions. PositiveCTL activation can be determined by assaying the culture for thepresence of CTLs that lyse radio-labeled target cells, either specificpeptide-pulsed targets or target cells that express endogenouslyprocessed antigen from which the specific peptide was derived.Alternatively, the presence of epitope-specific CTLs can be determinedby IFNγ in situ ELISA.

Additionally, a method has been devised which allows directquantification of antigen-specific T cells by staining withfluorescein-labelled HLA tetrameric complexes (Altman, J. D. et al.,Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et al., Science274:94, 1996). Other options include staining for intracellularlymphokines, and interferon release assays or ELISPOT assays. Tetramerstaining, intracellular lymphokine staining and ELISPOT assays allappear to be at least 10-fold more sensitive than more conventionalassays (Lalvani, A. et al., J. Exp. Med. 186:859, 1997; Dunbar, P. R. etal, Curr. Biol. 8:413, 1998; Murali-Krishna, K. et al., Immunity 8:177,1998).

Helper T lymphocyte (HTL) activation may also be assessed usingtechniques known to those in the art, such as T cell proliferation orlymphokine secretion (see, e.g. Alexander et al., Immunity 1:751-761,1994).

Alternatively, immunization of HLA transgenic mice can be used todetermine immunogenicity of peptide epitopes. Several transgenic mousestrains, e.g., mice with human A2.1, A11 (which can additionally be usedto analyze HLA-A3 epitopes), and B7 alleles have been characterized.Other transgenic mice strains (e.g., transgenic mice for HLA-A1 and A24)are being developed. Moreover, HLA-DR1 and HLA-DR3 mouse models havebeen developed. In accordance with principles in the art, additionaltransgenic mouse models with other HLA alleles are generated asnecessary.

Such mice can be immunized with peptides emulsified in IncompleteFreund's Adjuvant; thereafter any resulting T cells can be tested fortheir capacity to recognize target cells that have been peptide-pulsedor transfected with genes encoding the peptide of interest. CTLresponses can be analyzed using cytotoxicity assays described above.Similarly, HTL responses can be analyzed using, e.g., T cellproliferation or lymphokine secretion assays.

Vaccine Compositions

Vaccines that contain an immunologically effective amount of one or moreC35 peptide epitopes and/or C35 peptide epitope analogs of the inventionare a further embodiment of the invention. The peptides can be deliveredby various means or formulations, all collectively referred to as“vaccine” compositions. Such vaccine compositions, and/or modes ofadministration, can include, for example, naked cDNA in cationic lipidformulations; lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest.95:341, 1995), naked cDNA or peptides, encapsulated e.g., inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge,et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995); peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin ExpImmunol. 113:235-243, 1998); multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996); viral, bacterial, or,fungal delivery vectors (Perkus, M. E. et al., In: Concepts in vaccinedevelopment, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. etal., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986;Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al.,J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535,1990); particles of viral or synthetic origin (e.g., Kofler, N. et al.,J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol.30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995);adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev.Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993);liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L.,Immunol. Today 17:131, 1996); or, particle-absorbed cDNA (Ulmer, J. B.et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., andWebster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Conceptsin vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K.B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge,J. H. et al., Sem. Hematol. 30:16, 1993), etc. Toxin-targeted deliverytechnologies, also known as receptor mediated targeting, such as thoseof Avant Immunotherapeutics, Inc. (Needham, Mass.) or attached to astress protein, e.g., HSP 96 (Stressgen Biotechnologies Corp., Victoria,BC, Canada) can also be used.

Vaccines of the invention comprise nucleic acid mediated modalities. DNAor RNA encoding one or more of the polypeptides of the invention can beadministered to a patient. This approach is described, for instance, inWolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos.5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and,WO 98/04720. Examples of DNA-based delivery technologies include “nakedDNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery,cationic lipid complexes, and particle-mediated (“gene gun”) orpressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).Accordingly, peptide vaccines of the invention can be expressed by viralor bacterial vectors. Examples of expression vectors include attenuatedviral hosts, such as vaccinia or fowlpox. For example, vaccinia virus isused as a vector to express nucleotide sequences that encode thepeptides of the invention. Upon introduction into an acutely orchronically infected host or into a non-infected host, the recombinantvaccinia virus expresses the immunogenic peptide, and thereby elicits animmune response. Vaccinia vectors and methods useful in immunizationprotocols are described in, e.g., U.S. Pat. No. 4,722,848. Anothervector is BCG (Bacille Calmette Guerin). BCG vectors are described inStover et al., Nature 351:456-460 (1991). A wide variety of othervectors useful for therapeutic administration or immunization of thepeptides of the invention, e.g. adeno and adeno-associated virusvectors, alpha virus vectors, retroviral vectors, Salmonella typhivectors, detoxified anthrax toxin vectors, and the like, are apparent tothose skilled in the art from the description herein.

Furthermore, vaccines in accordance with the invention can comprise oneor more C35 peptide epitopes of the invention. Accordingly, a C35peptide epitope or C35 peptide epitope analog can be present in avaccine individually or; alternatively, the peptide epitope or analogcan exist as multiple copies of the same peptide epitope or analog (ahomopolymer), or as multiple different peptide epitopes or analogs (aheteropolymer). Polymers have the advantage of increased probability forimmunological reaction and, where different peptide epitopes or analogsare used to make up the polymer, the ability to induce antibodies and/orT cells that react with different antigenic determinants of the antigentargeted for an immune response. The composition may be a naturallyoccurring region of an antigen or can be prepared, e.g., recombinantlyor by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly L-lysine,poly L-glutamic acid, influenza virus proteins, hepatitis B virus coreprotein, and the like. The vaccines can contain a physiologicallytolerable diluent such as water, or a saline solution, preferablyphosphate buffered saline. Generally, the vaccines also include anadjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminumphosphate, aluminum hydroxide, or alum are examples of materials wellknown in the art. Additionally, as disclosed herein, CTL responses canbe primed by conjugating peptides of the invention to lipids, such astripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P₃CSS).

Upon immunization with a peptide composition in accordance with theinvention, via injection (e.g., subcutaneous, intradermal,intramuscular, aerosol, oral, transdermal, transmucosal, intrapleural,intrathecal), or other suitable routes, the immune system of the hostresponds to the vaccine by producing antibodies, CTLs and/or HTLsspecific for the desired antigen. Consequently, the host becomes atleast partially immune to subsequent exposure to the TAA, or at leastpartially resistant to further development of tumor associatedantigen-bearing cells and thereby derives a prophylactic or therapeuticbenefit.

In certain embodiments, components that induce T cell responses arecombined with components that induce antibody responses to the targetantigen of interest. A preferred embodiment of such a compositioncomprises class I and class II epitopes in accordance with theinvention. Alternatively, a composition comprises a class I and/or classII epitope in accordance with the invention, along with a PADRE™molecule (Epimmune, San Diego, Calif.).

Vaccine compositions of the invention can comprise antigen presentingcells, such as dendritic cells, as a vehicle to present peptides of theinvention. For example, dendritic cells are transfected, e.g., with aminigene construct in accordance with the invention, in order to elicitimmune responses. Minigenes are discussed in greater detail in afollowing section. Vaccine compositions can be created in vitro,following dendritic cell mobilization and harvesting, whereby loading ofdendritic cells occurs in vitro.

The vaccine compositions of the invention may also be used incombination with antiviral drugs such as interferon-α, immune adjuvantssuch as IL-12, GM-CSF, etc.

Preferably, the following principles are utilized when selectingepitope(s) for inclusion in a vaccine, either peptide-based or nucleicacid-based formulations. Each of the following principles can bebalanced in order to make the selection. When multiple epitopes are tobe used in a vaccine, the epitopes may be, but need not be, contiguousin sequence in the native antigen from which the epitopes are derived.

1) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with prevention orclearance of TAAexpressing tumors. For HLA Class I, this generallyincludes 3-4 epitopes derived from at least one TAA.

2) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, or for Class II an IC₅₀ of 1000 nM or less. ForHLA Class I it is presently preferred to select a peptide having an IC₅₀of 200 nM or less, as this is believed to better correlate not only toinduction of an immune response, but to in vitro tumor cell killing aswell.

3) Supermotif bearing-peptides, or a sufficient array of allele-specificmotif-bearing peptides, are selected to give broad population coverage.In general, it is preferable to have at least 80% population coverage. AMonte Carlo analysis, a statistical evaluation known in the art, can beemployed to assess the breadth of population coverage.

4) When selecting epitopes from cancer-related antigens, it can bepreferable to include analog peptides in the selection, because thepatient may have developed tolerance to the native epitope. Whenselecting epitopes for infectious disease-related antigens it ispresently preferable to select either native or analog epitopes.

5) Of particular relevance are “nested epitopes.” Nested epitopes (orepitope analogs) occur where at least two epitopes or analogs (or anepitope and an analog) overlap in a given polypeptide sequence. Apolypeptide comprising “transcendent nested epitopes” is a polypeptidethat has both HLA class I and HLA class II epitopes and/or analogs init. When providing nested epitopes, it is preferable to provide asequence that has the greatest number of epitopes or analogs perprovided sequence. Preferably, one avoids providing a polypeptide thatis any longer than the combined length of the peptide epitopes oranalogs. When providing a polypeptide comprising nested epitopes, it isimportant to evaluate the polypeptide in order to insure that it doesnot have pathological or other deleterious biological properties; thisis particularly relevant for vaccines directed to infectious organisms.Thus, in a preferred embodiment, the vaccine compositions of theinvention comprise one or more multi-epitope polypeptides selected fromthe group consisting of: I105 to V113 of SEQ ID NO:2 and FIG. 1B, T101to V113 of SEQ ID NO:2 and FIG. 1B, E100 to V113 of SEQ ID NO:2 and FIG.1B, G99 to V113 of SEQ ID NO:2 and FIG. 1B, I93 to V113 of SEQ ID NO:2and FIG. 1B, D88 to V113 of SEQ ID NO:2 and FIG. 1B, P84 to V113 of SEQID NO:2 and FIG. 1B, K77 to V113 of SEQ ID NO:2 and FIG. 1B, Q72 to V113of SEQ ID NO:2 and FIG. 1B, F65 to V113 of SEQ ID NO:2 and FIG. 1B, andL59 to V113 of SEQ ID NO:2 and FIG. 1B.

6) If a polypeptide comprising more than one C35 peptide epitope or C35peptide epitope analog is created, or when creating a minigene, anobjective is to generate the smallest polypeptide that encompasses theepitopes/analogs of interest. This principle is similar, if not the sameas that employed when selecting a polypeptide comprising nestedepitopes. However, with an artificial polyepitopic polypeptide, the sizeminimization objective is balanced against the need to integrate anyspacer sequences between epitopes in the polyepitopic polypeptide.Spacer or linker amino acid residues can be introduced to avoidjunctional epitopes (an epitope recognized by the immune system, notpresent in the target antigen, and only created by the man-madejuxtaposition of epitopes), or to facilitate cleavage between epitopesand thereby enhance epitope presentation. Junctional epitopes aregenerally to be avoided because the recipient may generate an immuneresponse to that non-native epitope. Of particular concern is ajunctional epitope that is a “dominant epitope.” A dominant epitope maylead to such a zealous response that immune responses to other epitopesare diminished or suppressed.

Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding multiple C35peptide epitopes or analogs are a useful embodiment of the invention;discrete epitopes/analogs or polyepitopic polypeptides can be encoded.The epitopes or analogs to be included in a minigene are preferablyselected according to the guidelines set forth in the previous section.Examples of amino acid sequences that can be included in a minigeneinclude: HLA class I epitopes or analogs, HLA class II epitopes oranalogs, a ubiquitination signal sequence, and/or a targeting sequencesuch as an endoplasmic reticulum (ER) signal sequence to facilitatemovement of the resulting peptide into the endoplasmic reticulum.

The use of multi-epitope minigenes is also described in, e.g., Ishiokaet al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. A similar approach can be used to develop minigenesencoding TAA epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. However, tooptimize expression and/or immunogenicity, additional elements can beincorporated into the minigene design such as one or more spacer orlinker amino acid residues between epitopes. HLA presentation of CTL andHTL epitopes may be improved by including synthetic (e.g. poly-alanine)or naturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger polypeptides comprising the epitope(s)/analog(s)are within the scope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adownstream cloning site for minigene insertion; a polyadenylation signalfor efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Optimized peptide expression and immunogenicity can be achieved bycertain modifications to a minigene construct. For example, in somecases introns facilitate efficient gene expression, thus one or moresynthetic or naturally-occurring introns can be incorporated into thetranscribed region of the minigene. The inclusion of mRNA stabilizationsequences and sequences for replication in mammalian cells may also beconsidered for increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate bacterial strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as cell banks.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence to enhanceimmunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(e.g., one that modulates immunogenicity) can be used. Examples ofproteins or polypeptides that, if co-expressed with epitopes, canenhance an immune response include cytokines (e.g., IL-2, IL-12, GMCSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orpan-DR binding proteins (PADRE™, Epimmune, San Diego, Calif.). Helper Tcell (HTL) epitopes such as PADRE™ molecules can be joined tointracellular targeting signals and expressed separately from expressedCTL epitopes. This can be done in order to direct HTL epitopes to a cellcompartment different than that of the CTL epitopes, one that providesfor more efficient entry of HTL epitopes into the HLA class II pathway,thereby improving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and are grown tosaturation in shaker flasks or a bioreactor according to well knowntechniques. Plasmid DNA is purified using standard bioseparationtechnologies such as solid phase anion-exchange resins available, e.g.,from QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA canbe isolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigenevaccines, alternative methods of formulating purified plasmid DNA may beused. A variety of such methods have been described, and new techniquesmay become available. Cationic lipids, glycolipids, and fusogenicliposomes can also be used in the formulation (see, e.g., WO 93/24640;Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No.5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA84:7413 (1987). In addition, peptides and compounds referred tocollectively as protective, interactive, non-condensing compounds (PINC)can also be complexed to purified plasmid DNA to influence variablessuch as stability, intramuscular dispersion, or trafficking to specificorgans or cell types.

Target cell sensitization can be used as a functional assay of theexpression and HLA class I presentation of minigene-encoded epitopes.For example, the plasmid DNA is introduced into a mammalian cell linethat is a suitable target for standard CTL chromium release assays. Thetransfection method used will be dependent on the final formulation,electroporation can be used for “naked” DNA, whereas cationic lipidsallow direct in vitro transfection. A plasmid expressing greenfluorescent protein (GFP) can be co-transfected to allow enrichment oftransfected cells using fluorescence activated cell sorting (FACS). Thetransfected cells are then chromium-51 (⁵¹Cr) labeled and used astargets for epitope-specific CTLs. Cytolysis of the target cells,detected by ⁵¹Cr release, indicates both the production and HLApresentation of, minigene-encoded CTL epitopes. Expression of HTLepitopes may be evaluated in an analogous manner using assays to assessHTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (IP) for lipid-complexed DNA). Eleven to twenty-one daysafter immunization, splenocytes are harvested and restimulated for oneweek in the presence of peptides encoding each epitope being tested.Thereafter, for CTLs, standard assays are conducted to determine ifthere is cytolysis of peptide-loaded, ⁵¹Cr-labeled target cells. Onceagain, lysis of target cells that were exposed to epitopes correspondingto those in the minigene, demonstrates DNA vaccine function andinduction of CTLs. Immunogenicity of HTL epitopes is evaluated intransgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment for ballistic delivery, DNA can beadhered to particles, such as gold particles.

Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the present inventioncan be modified to provide desired attributes, such as improved serumhalf-life, broadened population coverage or enhanced immunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the CTL peptide to a sequence which contains atleast one HTL epitope.

Although a CTL peptide can be directly linked to a T helper peptide,particularly preferred CTL epitope/HTL epitope conjugates are linked bya spacer molecule. The spacer is typically comprised of relativelysmall, neutral molecules, e.g., amino acids or amino acid mimetics,which are substantially uncharged under physiological conditions. Thespacers are typically selected from, e.g., Ala, Gly, or other neutralspacers of nonpolar amino acids or neutral polar amino acids. It will beunderstood that the optional spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, commonly three to13, more frequently three to six residues. The CTL peptide epitope maybe linked to the T helper peptide epitope, directly or via a spacer, ateither it's amino or carboxyl terminus. The amino terminus of either theCTL peptide or the HTL peptide can be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in the majority of the population. This can beaccomplished by selecting amino acid sequences that bind to many, most,or all of the HLA class II molecules. These are known as “looselyHLA-restricted” or “promiscuous” T helper sequences. Examples of aminoacid sequences that are promiscuous include sequences from antigens suchas tetanus toxoid at positions 830-843 (QYIKANSKFIGITE (SEQ IDNO:2055)), Plasmodium falciparum CS protein at positions 378-398(DIEKKIAKMEKASSVFNVVNS (SEQ ID NO:2056)), and Streptococcus 18 kDprotein at positions 116 (GAVDSILGGVATYGAA (SEQ ID NO:2057)). Otherexamples include peptides bearing a DR 1-4-7 supermotif, or either ofthe DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences that may not be found in nature. Syntheticcompounds fall within the family of molecules called Pan-DR-bindingepitopes (e.g., PADRE™, Epimmune Inc., San Diego, Calif.). PADRE™peptides are designed to bind multiple HLA-DR (human HLA class II)molecules. For instance, a pan-DR-binding epitope peptide having theformula: aKXVAAZTLKAAa (SEQ ID NO:2058), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine; “Z” is either tryptophan,tyrosine, histidine or asparagine; and “a” is either D-alanine orL-alanine, has been found to bind to numerous allele-specific HLA-DRmolecules. Accordingly, these molecules stimulate a T helper lymphocyteresponse from most individuals, regardless of their HLA type. Certainpan-DR binding epitopes comprise all “L” natural amino acids; thesemolecules can be provided as peptides or in the form of nucleic acidsthat encode the peptide.

HTL peptide epitopes can be modified to alter their biologicalproperties. HTL peptide epitopes can be modified in the same manner asCTL peptides. For instance, they may be modified to include D-aminoacids or be conjugated to other molecules such as lipids, proteins,sugars and the like. Peptides comprising D-amino acids generally haveincreased resistance to proteases, and thus have an extended serumhalf-life.

In addition, polypeptides comprising one or more peptide epitopes of theinvention can be conjugated to other molecules such as lipids, proteinsor sugars, or any other synthetic compounds, to increase theirbiological activity. For example, a T helper peptide can be conjugatedto one or more palmitic acid chains at either the amino or the carboxyltermini.

Combinations of CTL Peptides with T Cell Priming Materials

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primescytotoxic T lymphocytes. Lipids have been identified as agents capableof facilitating the priming in vitro CTL response against viralantigens. For example, palmitic acid residues can be attached to the ε-and α-amino groups of a lysine residue and then linked to an immunogenicpeptide. One or more linking moieties can be used such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like. The lipidated peptide can then beadministered directly in a micelle or particle, incorporated into aliposome, or emulsified in an adjuvant, e.g., incomplete Freund'sadjuvant. A preferred immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys via a linking moiety, e.g.,Ser-Ser, added to the amino terminus of an immunogenic peptide.

In another embodiment of lipid-facilitated priming of CTL responses, E.coli lipoproteins, such astripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P₃CSS) can be used toprime CTL when covalently attached to an appropriate peptide. (See,e.g., Deres, et al., Nature 342:561, 1989). Thus, peptides of theinvention can be coupled to P₃CSS, and the lipopeptide administered toan individual to specifically prime a CTL response to the targetantigen. Moreover, because the induction of neutralizing antibodies canalso be primed with P₃CSS-conjugated epitopes, two such compositions canbe combined to elicit both humoral and cell-mediated responses.

Vaccine Compositions Comprising Dendritic Cells Pulsed with CTL and/orHTL Peptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asPROGENIPOIETIN™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsingthe DC with peptides and prior to reinfusion into patients, the DC arewashed to remove unbound peptides. In this embodiment, a vaccinecomprises peptide-pulsed DCs which present the pulsed peptide epitopesin HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to one or more antigens of interest, e.g., tumorassociated antigens (TAA) such as HER2/neu, p53, MAGE 2, MAGE3, and/orcarcinoembryonic antigen (CEA). Collectively, these TAA are associatedwith breast, colon and lung cancers. Optionally, a helper T cell (HTL)peptide such as PADRE™, can be included to facilitate the CTL response.Thus, a vaccine in accordance with the invention comprising epitopesfrom HER2/neu, p53, MAGE2, MAGE3, and carcinoembryonic antigen (CEA) isused to treat minimal or residual disease in patients with malignanciessuch as breast, colon or lung cancer; any malignancies that bear any ofthese TAAs can also be treated with the vaccine. A TAA vaccine can beused following debulking procedures such as surgery, radiation therapyor chemotherapy, whereupon the vaccine provides the benefit ofincreasing disease free survival and overall survival in the recipients.

Thus, in preferred embodiments, a vaccine of the invention is a productthat treats a majority of patients across a number of different tumortypes. A vaccine comprising a plurality of epitopes, preferablysupermotif-bearing epitopes, offers such an advantage.

Administration of Vaccines for Therapeutic or Prophylactic Purposes

The polypeptides comprising one or more peptide epitopes of the presentinvention, including pharmaceutical and vaccine compositions thereof,are useful for administration to mammals, particularly humans, to treatand/or prevent disease. In one embodiment, vaccine compositions (peptideor nucleic acid) of the invention are administered to a patient who hasa malignancy associated with expression of one or more TAAs, or to anindividual susceptible to, or otherwise at risk for developingTAA-related disease. Upon administration an immune response is elicitedagainst the TAAs, thereby enhancing the patient's own immune responsecapabilities. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective immune response to the TAA-expressing cells and tothereby cure, arrest or slow symptoms and/or complications. An amountadequate to accomplish this is defined as “therapeutically effectivedose.” Amounts effective for this use will depend on, e.g. theparticular composition administered, the manner of administration, thestage and severity of the disease being treated, the weight and generalstate of health of the patient, and the judgment of the prescribingphysician.

The vaccine compositions of the invention can be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg of peptide and the higher valueis about 10,000; 20,000; 30,000; or 50,000 μg of peptide. Dosage valuesfor a human typically range from about 500 μg to about 50,000 μg ofpeptide per 70 kilogram patient. This is followed by boosting dosages ofbetween about 1.0 μg to about 50,000 μg of peptide, administered atdefined intervals from about four weeks to six months after the initialadministration of vaccine. The immunogenicity of the vaccine may beassessed by measuring the specific activity of CTL and HTL obtained froma sample of the patient's blood.

As noted above, polypeptides comprising CTL and/or HTL epitopes of theinvention induce immune responses when presented by HLA molecules andcontacted with a CTL or HTL specific for an epitope comprised by thepeptide. The manner in which the peptide is contacted with the CTL orHTL is not critical to the invention. For instance, the peptide can becontacted with the CTL or HTL either in vitro or in vivo. If thecontacting occurs in vivo, peptide can be administered directly, or inother forms/vehicles, e.g., DNA vectors encoding one or more peptides,viral vectors encoding the peptide(s), liposomes, antigen presentingcells such as dendritic cells, and the like, as described herein.

Accordingly, for pharmaceutical compositions of the invention in theform of peptides or polypeptides, the peptides or polypeptides can beadministered directly. Alternatively, the peptide/polypeptides can beadministered indirectly presented on APCs, or as DNA encoding them.Furthermore, the polypeptides, peptide epitopes or DNA encoding them canbe administered individually or as fusions of one or more peptidesequences.

For therapeutic use, administration should generally begin at the firstdiagnosis of TAA-related disease. This is followed by boosting doses atleast until symptoms are substantially abated and for a periodthereafter. In chronic disease states, loading doses followed byboosting doses may be required.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg of peptide and the higher value is about 10,000; 20,000; 30,000; or50,000 μg of peptide. Dosage values for a human typically range fromabout 500 μg to about 50,000 μg of peptide per 70 kilogram patient.Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide,administered pursuant to a boosting regimen over weeks to months, can beadministered depending upon the patient's response and condition.Patient response can be determined by measuring the specific activity ofCTL and HTL obtained from the patient's blood.

In certain embodiments, polypeptides, peptides and compositions of thepresent invention are used in serious disease states. In such cases, asa result of the minimal amounts of extraneous substances and therelative nontoxic nature of the peptides, it is possible and may bedesirable to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

For treatment of chronic disease, a representative dose is in the rangedisclosed above, namely where the lower value is about 1, 5, 50, 500, or1,000 μg of peptide and the higher value is about 10,000; 20,000;30,000; or 50,000 μg of peptide, preferably from about 500 μg to about50,000 μg of peptide per 70 kilogram patient. Initial doses followed byboosting doses at established intervals, e.g., from four weeks to sixmonths, may be required, possibly for a prolonged period of time toeffectively immunize an individual. In the case of chronic disease,administration should continue until at least clinical symptoms orlaboratory tests indicate that the disease has been eliminated orsubstantially abated, and for a follow-up period thereafter. Thedosages, routes of administration, and dose schedules are adjusted inaccordance with methodologies known in the art.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, intrathecal, or local administration.Preferably, the pharmaceutical compositions are administered parentally,e.g., intravenously, subcutaneously, intradermally, or intramuscularly.

Thus, in a preferred embodiment the invention provides compositions forparenteral administration which comprise a solution of the immunogenicpeptides dissolved or suspended in an acceptable carrier, preferably anaqueous carrier. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like.These compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances or pharmaceutical excipients as may be required toapproximate physiological conditions, such as pH-adjusting and bufferingagents, tonicity adjusting agents, wetting agents, preservatives, andthe like, for example, sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc.

The concentration of peptides and polypeptides of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

A human unit dose form of the peptide and polypeptide composition istypically included in a pharmaceutical composition that also comprises ahuman unit dose of an acceptable carrier, preferably an aqueous carrier,and is administered in a volume of fluid that is known by those of skillin the art to be used for administration of such compositions to humans(see, e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A.Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985).

The peptides and polypeptides of the invention can also be administeredvia liposomes, which serve to target the peptides and polypeptides to aparticular tissue, such as lymphoid tissue, or to target selectively toinfected cells, as well as to increase the half-life of the peptidecomposition. Liposomes include emulsions, foams, micelles, insolublemonolayers, liquid crystals, phospholipid dispersions, lamellar layersand the like. In these preparations, the peptides and polypeptides to bedelivered is incorporated as part of a liposome, alone or in conjunctionwith a molecule which binds to a receptor prevalent among lymphoid cells(such as monoclonal antibodies which bind to the CD45 antigen) or withother therapeutic or immunogenic compositions. Thus, liposomes eitherfilled or decorated with a desired peptide of the invention can bedirected to the site of lymphoid cells, where the liposomes then deliverthe peptide compositions. Liposomes for use in accordance with theinvention are formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of, e.g., liposome size, acid lability and stability ofthe liposomes in the blood stream. A variety of methods are availablefor preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev.Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728,4,837,028, and 5,019,369.

For targeting compositions of the invention to cells of the immunesystem, a ligand can be incorporated into the liposome, e.g., antibodiesor fragments thereof specific for cell surface determinants of thedesired immune system cells. A liposome suspension containing a peptidemay be administered intravenously, locally, topically, etc. in a dosewhich varies according to, inter alia, the manner of administration, thepeptide being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, often at aconcentration of 25%-75%.

For aerosol administration, the immunogenic peptides are preferablysupplied in finely divided form, along with a surfactant and propellant.Typical percentages of peptides are 0.01%-20% by weight, often 1%-10%.The surfactant must, of course, be pharmaceutically acceptable, andpreferably soluble in the propellant. Representative of such agents arethe esters or partial esters of fatty acids containing from 6 to 22carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,linoleic, linolenic, olesteric and oleic acids with an aliphaticpolyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixedor natural glycerides may be employed. The surfactant may constitute0.1%-20% by weight of the composition, preferably 0.25-5%. The balanceof the composition is ordinarily propellant, although an atomizer may beused in which no propellant is necessary and other percentages areadjusted accordingly. A carrier can also be included, e.g., lecithin forintranasal delivery.

Antigenic peptides of the invention have been used to elicit a CTLand/or HTL response ex vivo, as well. The resulting CTLs or HTLs can beused to treat chronic infections, or tumors in patients that do notrespond to other conventional forms of therapy, or who do not respond toa therapeutic peptide or nucleic acid vaccine in accordance with theinvention. Ex vivo CTL or HTL responses to a particular antigen(infectious or tumor-associated) are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (aninfected cell or a tumor cell).

A number of computer algorithms have been described for identificationof peptides in a larger protein that satisfy the requirements of peptidebinding motifs for specific MHC class I or MHC class II molecules.Because of the extensive polymorphism of MHC molecules, differentpeptides will often bind to different MHC molecules. Tables 1-6 list C35peptides predicted to be MHC binding peptides using three differentalgorithms. Specifically, Tables 1 and 5 list C35 HLA Class I and IIepitopes predicted using the rules found at the SYFPEITHI website(wysiwyg://35/http://134.2.96.221/scripts/hlaserver.dll/EpPredict.htm)and are based on the book “MHC Ligands and Peptide Motifs” by Rammensee,H. G., Bachmann, J. and Stevanovic, S. (Chapman & Hall, New York 1997).Table 2 lists predicted MHC binding peptides derived from the C35sequence using the NIH BIMAS program available on the web(bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform). Finally,Tables 3 and 6 list predicted C35 peptides identified by the Tepitopeprogram, a program for prediction of peptides that may bind to multipledifferent MHC class II molecules. Using Tepitope, four C35 peptides wereidentified as likely candidates for binding to a variety of HLA class IImolecules. These peptides are, in general, longer than those binding toHLA class I and more degenerate in terms of binding to multiple HLAclass II molecules. Due to the relatedness of the HLA molecules and theinherent limitations of the binding algorithms, it is expected that manyof these C35 peptide epitopes predicted to bind to a specific HLAmolecules will also bind to one or more other HLA molecules.

TABLE 1 C35 peptides predicted by SYFPEITHI website(score reflects ligation strength): Class I MHC HLA-A*0201 nonamersSEQ ID NO: 2 Start Position (“Pos”) 1 2 3 4 5 6 7 8 9 Score  9 S V A P PP E E V 23 88 D L I E A I R R A 21 37 A T Y L E L A S A 19 97 S N G E TL E K I 18 105 I T N S R P P C V 18  2 S G E P G Q T S V 17 45 A V K E QY P G I 17 38 T Y L E L A S A V 16 61 G T G A F E I E I 16 85 Y E K D LI E A I 16 65 F E I E I N G Q L 15 107  N S R P P C V I L 15 41 E L A SA V K E Q 14 58 R L G G T G A F E 14 59 L G G T G A F E I 14 66 E I E IN G Q L V 14 68 E I N G Q L V F S 14 81 G G F P Y E K D L 14 94 R R A SN G E T L 14 HLA-A*0201 decamers Pos 1 2 3 4 5 6 7 8 9 0 Score 58 R L GG T G A F E I 22 96 A S N G E T L E K I 19 104  K I T N S R P P C V 1937 A T Y L E L A S A V 18 17 V E P G S G V R I V 17 33 C G F E A T Y L EL 16 44 S A V K E Q Y P G I 16 92 A I R R A S N G E T 16 39 Y L E L A SA V K E 15 53 I E I E S R L G G T 15 65 F E I E I N G Q L V 15 105  I TN S R P P C V I 15 1 M S G E P G Q T S V 14 63 G A F E I E I N G Q 14 68E I N G Q L V F S K 14 69 I N G Q L V F S K L 14 83 F P Y E K D L I E A14 88 D L I E A I R R A S 14 93 I R R A S N G E T L 14 72 Q L V F S K LE N G 13 89 L I E A I R R A S N 13  8 T S V A P P P E E V 12 16 E V E PG S G V R I 12 50 Y P G I E I E S R L 12 60 G G T G A F E I E I 12 81 GG F P Y E K D L I 12 106  T N S R P P C V I L 12 HLA-A*0203 nonamers Pos1 2 3 4 5 6 7 8 9 Score 35 F E A T Y L E L A 12 HLA-A*0203 decamers Pos1 2 3 4 5 6 7 8 9 0 Score 36 E A T Y L E L A S A 18 HLA-A1 nonamers Pos1 2 3 4 5 6 7 8 9 Score 77 K L E N G G F P Y 29 2 S G E P G Q T S V 1821 S G V R I V V E Y 18 16 E V E P G S G V R 17 29 Y C E P C G F E A 1742 L A S A V K E Q Y 17 31 E P C G F E A T Y 16 34 G F E A T Y L E L 1639 Y L E L A S A V K 14 84 P Y E K D L I E A 14 66 E I E I N G Q L V 1313 P P E E V E P G S 12 46 V K E Q Y P G I E 12 52 G I E I E S R L G 1296 A S N G E T L E K 12 HLA-A1 decamers Pos 1 2 3 4 5 6 7 8 9 0 Score 20G S G V R I V V E Y 20 29 Y C E P C G F E A T 19 76 S K L E N G G F P Y18 2 S G E P G Q T S V A 17 52 G I E I E S R L G G 17 66 E I E I N G Q LV F 17 41 E L A S A V K E Q Y 16 46 V K E Q Y P G I E I 16 16 E V E P GS G V R I 15 30 C E P C G F E A T Y 15 39 Y L E L A S A V K E 15 77 K LE N G G F P Y E 14 86 E K D L I E A I R R 14 98 N G E T L E K I T N 1434 G F E A T Y L E L A 12 64 A F E I E I N G Q L 12 101 T L E K I T N SR P 12 HLA-A26 nonamers Pos 1 2 3 4 5 6 7 8 9 Score 68 E I N G Q L V F S24 100  E T L E K I T N S 24 88 D L I E A I R R A 23 54 E I E S R L G GT 22 41 E L A S A V K E Q 21 45 A V K E Q Y P G I 20 31 E P C G F E A TY 19 34 G F E A T Y L E L 19 73 L V F S K L E N G 19 16 E V E P G S G VR 18 77 K L E N G G F P Y 18 66 E I E I N G Q L V 17 21 S G V R I V V EY 16 37 A T Y L E L A S A 16 24 R I V V E Y C E P 15  9 S V A P P P E EV 14 22 G V R I V V E Y C 14 51 P G I E I E S R L 14 70 N G Q L V F S KL 14 57 S R L G G T G A F 13 65 F E I E I N G Q L 13 25 I V V E Y C E PC 12 48 E Q Y P G I E I E 12 67 I E I N G Q L V F 12 75 F S K L E N G GF 12 81 G G F P Y E K D L 12 104  K I T N S R P P C 12 105  I T N S R PP C V 12 HLA-A26 decamers Pos 1 2 3 4 5 6 7 8 9 0 Score 41 E L A S A V KE Q Y 27 66 E I E I N G Q L V F 26 68 E I N G Q L V F S K 23 26 V V E YC E P C G F 21 16 E V E P G S G V R I 20 88 D L I E A I R R A S 19 100 E T L E K I T N S R 19 74 V F S K L E N G G F 18 33 C G F E A T Y L E L17 54 E I E S R L G G T G 17 56 E S R L G G T G A F 17 20 G S G V R I VV E Y 16 31 E P C G F E A T Y L 16 64 A F E I E I N G Q L 15 69 I N G QL V F S K L 15 61 G T G A F E I E I N 14 73 L V F S K L E N G G 14  9 SV A P P P E E V E 13 25 I V V E Y C E P C G 13 45 A V K E Q Y P G I E 1372 Q L V F S K L E N G 13 77 K L E N G G F P Y E 13 79 E N G G F P Y E KD 13  4 E P G Q T S V A P P 12  7 Q T S V A P P P E E 12 30 C E P C G FE A T Y 12 36 E A T Y L E L A S A 12 37 A T Y L E L A S A V 12 76 S K LE N G G F P Y 12 89 L I E A I R R A S N 12 HLA-A3 nonamers Pos 1 2 3 4 56 7 8 9 Score 39 Y L E L A S A V K 28 77 K L E N G G F P Y 25 16 E V E PG S G V R 24 58 R L G G T G A F E 22 67 I E I N G Q L V F 19 96 A S N GE T L E K 18 92 A I R R A S N G E 17  9 S V A P P P E E V 16 101  T L EK I T N S R 16 22 G V R I V V E Y C 15 31 E P C G F E A T Y 15 45 A V KE Q Y P G I 15 72 Q L V F S K L E N 15 21 S G V R I V V E Y 14 68 E I NG Q L V F S 14 69 I N G Q L V F S K 14 88 D L I E A I R R A 14 91 E A IR R A S N G 14 25 I V V E Y C E P C 13 37 A T Y L E L A S A 13 55 I E SR L G G T G 13 57 S R L G G T G A F 13 79 E N G G F P Y E K 13 87 K D LI E A I R R 13 104  K I T N S R P P C 13 24 R I V V E Y C E P 12 42 L AS A V K E Q Y 12 66 E I E I N G Q L V 12 89 L I E A I R R A S 12 90 I EA I R R A S N 12 94 R R A S N G E T L 12 HLA-A3 decamers Pos 1 2 3 4 5 67 8 9 0 Score 68 E I N G Q L V F S K 22 16 E V E P G S G V R I 20 38 T YL E L A S A V K 20 41 E L A S A V K E Q Y 20 66 E I E I N G Q L V F 20 9 S V A P P P E E V E 19 58 R L G G T G A F E I 19 39 Y L E L A S A V KE 18 92 A I R R A S N G E T 18 95 R A S N G E T L E K 18 45 A V K E Q YP G I E 17 54 E I E S R L G G T G 16 88 D L I E A I R R A S 16 89 L I EA I R R A S N 16 26 V V E Y C E P C G F 15 37 A T Y L E L A S A V 15 22G V R I V V E Y C E 14 77 K L E N G G F P Y E 14 93 I R R A S N G E T L14 25 I V V E Y C E P C G 13 30 C E P C G F E A T Y 13 52 G I E I E S RL G G 13 76 S K L E N G G F P Y 13 78 L E N G G F P Y E K 13 101  T L EK I T N S R P 13 104  K I T N S R P P C V 13 24 R I V V E Y C E P C 1272 Q L V F S K L E N G 12 HLA-B*0702 nonamers Pos 1 2 3 4 5 6 7 8 9Score 18 E P G S G V R I V 19 107  N S R P P C V I L 18  4 E P G Q T S VA P 15 11 A P P P E E V E P 15 31 E P C G F E A T Y 14 34 G F E A T Y LE L 13 94 R R A S N G E T L 13 12 P P P E E V E P G 12 19 P G S G V R IV V 12 32 P C G F E A T Y L 12 83 F P Y E K D L I E 12 106  T N S R P PC V I 12 HLA-B*0702 decamers Pos 1 2 3 4 5 6 7 8 9 0 Score 31 E P C G FE A T Y L 24 50 Y P G I E I E S R L 21 18 E P G S G V R I V V 20 83 F PY E K D L I E A 16 4 E P G Q T S V A P P 15 11 A P P P E E V E P G 15 93I R R A S N G E T L 14 106  T N S R P P C V I L 14 69 I N G Q L V F S KL 13 33 C G F E A T Y L E L 12 64 A F E I E I N G Q L 12HLA-B*08 octamers Pos 1 2 3 4 5 6 7 8 Score 83 F P Y E K D L I 25 66 E IE I N G Q L 16 52 G I E I E S R L 15 18 E P G S G V R I 14 54 E I E S RL G G 14 91 E A I R R A S N 14 95 R A S N G E T L 14 100  E T L E K I TN 14 33 C G F E A T Y L 12 45 A V K E Q Y P G 12 58 R L G G T G A F 1268 E I N G Q L V F 12 71 G Q L V F S K L 12 75 F S K L E N G G 12 82 G FP Y E K D L 12 107  N S R P P C V I 12 108  S R P P C V I L 12HLA-B*08 nonamers Pos 1 2 3 4 5 6 7 8 9 Score 75 F S K L E N G G F 19 83F P Y E K D L I E 19 45 A V K E Q Y P G I 18 85 Y E K D L I E A I 18107  N S R P P C V I L 17 100  E T L E K I T N S 15 54 E I E S R L G G T14 65 F E I E I N G Q L 14 91 E A I R R A S N G 14 20 G S G V R I V V E12 34 G F E A T Y L E L 12 51 P G I E I E S R L 12 81 G G F P Y E K D L12 HLA-B*1510 nonamers Pos 1 2 3 4 5 6 7 8 9 Score 107  N S R P P C V IL 15 34 G F E A T Y L E L 13 51 P G I E I E S R L 13 81 G G F P Y E K DL 13 94 R R A S N G E T L 13 HLA-B*2705 nonamers Pos 1 2 3 4 5 6 7 8 9Score 57 S R L G G T G A F 26 94 R R A S N G E T L 25 67 I E I N G Q L VF 19 87 K D L I E A I R R 19 51 P G I E I E S R L 17 81 G G F P Y E K DL 17 65 F E I E I N G Q L 16 69 I N G Q L V F S K 16 96 A S N G E T L EK 16 16 E V E P G S G V R 15 34 G F E A T Y L E L 15 50 Y P G I E I E SR 15 70 N G Q L V F S K L 15 101  T L E K I T N S R 15 23 V R I V V E YC E 14 32 P C G F E A T Y L 14 39 Y L E L A S A V K 14 79 E N G G F P YE K 14 93 I R R A S N G E T 14 21 S G V R I V V E Y 13 27 V E Y C E P CG F 13 75 F S K L E N G G F 13 86 E K D L I E A I R 13 107  N S R P P CV I L 13 17 V E P G S G V R I 12 31 E P C G F E A T Y 12 77 K L E N G GF P Y 12 HLA-B*2709 nonamers Pos 1 2 3 4 5 6 7 8 9 Score 94 R R A S N GE T L 25 57 S R L G G T G A F 20 81 G G F P Y E K D L 16 34 G F E A T YL E L 14 51 P G I E I E S R L 13 65 F E I E I N G Q L 13 23 V R I V V EY C E 12 107  N S R P P C V I L 12 HLA-B*5101 nonamers Pos 1 2 3 4 5 6 78 9 Score 18 E P G S G V R I V 21 81 G G F P Y E K D L 21 51 P G I E I ES R L 20 70 N G Q L V F S K L 20 19 P G S G V R I V V 19 31 E P C G F EA T Y 19  2 S G E P G Q T S V 18 42 L A S A V K E Q Y 18 59 L G G T G AF E I 18 21 S G V R I V V E Y 14 83 F P Y E K D L I E 14 97 S N G E T LE K I 14 13 P P E E V E P G S 13 38 T Y L E L A S A V 13 45 A V K E Q YP G I 13 63 G A F E I E I N G 13 94 R R A S N G E T L 13 12 P P P E E VE P G 12 33 C G F E A T Y L E 12 50 Y P G I E I E S R 12 66 E I E I N GQ L V 12 85 Y E K D L I E A I 12 95 R A S N G E T L E 12 105  I T N S RP P C V 12 HLA-B*5101 octamers Pos 1 2 3 4 5 6 7 8 Score 83 F P Y E K DL I 25 95 R A S N G E T L 23 10 V A P P P E E V 21 18 E P G S G V R I 2133 C G F E A T Y L 21 98 N G E T L E K I 19 19 P G S G V R I V 18 60 G GT G A F E I 18 62 T G A F E I E I 18 63 G A F E I E I N 14 71 G Q L V FS K L 14 48 E Q Y P G I E I 13 67 I E I N G Q L V 13 106  T N S R P P CV 12 Class II MHC HLA-DRB1*0101 15-mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 45 Score 72 Q L V F S K L E N G G F P Y E 29 37 A T Y L E L A S A V K E QY P 26 26 V V E Y C E P C G F E A T Y L 25 63 G A F E I E I N G Q L V FS K 25 24 R I V V E Y C E P C G F E A T 24 36 E A T Y L E L A S A V K EQ Y 24 39 Y L E L A S A V K E Q Y P G I 24 53 I E I E S R L G G T G A FE I 24 56 E S R L G G T G A F E I E I N 24 14 P E E V E P G S G V R I VV E 23 43 A S A V K E Q Y P G I E I E S 23 20 G S G V R I V V E Y C E PC G 20 62 T G A F E I E I N G Q L V F S 20 32 P C G F E A T Y L E L A SA V 19 47 K E Q Y P G I E I E S R L G G 19 64 A F E I E I N G Q L V F SK L 19 82 G F P Y E K D L I E A I R R A 19 34 G F E A T Y L E L A S A VK E 18 54 E I E S R L G G T G A F E I E 18 90 I E A I R R A S N G E T LE K 18 99 G E T L E K I T N S R P P C V 18 31 E P C G F E A T Y L E L AS A 17 49 Q Y P G I E I E S R L G G T G 17 58 R L G G T G A F E I E I NG Q 17 66 E I E I N G Q L V F S K L E N 17 67 I E I N G Q L V F S K L EN G 17 68 E I N G Q L V F S K L E N G G 17 84 P Y E K D L I E A I R R AS N 17 86 E K D L I E A I R R A S N G E 17 35 F E A T Y L E L A S A V KE Q 16 74 V F S K L E N G G F P Y E K D 16 87 K D L I E A I R R A S N GE T 16 91 E A I R R A S N G E T L E K I 16  1 M S G E P G Q T S V A P PP E 15  4 E P G Q T S V A P P P E E V E 15 11 A P P P E E V E P G S G VR I 15 12 P P P E E V E P G S G V R I V 15 29 Y C E P C G F E A T Y L EL A 15  5 P G Q T S V A P P P E E V E P 14  6 G Q T S V A P P P E E V EP G 14 44 S A V K E Q Y P G I E I E S R 14 52 G I E I E S R L G G T G AF E 14 61 G T G A F E I E I N G Q L V F 13 50 Y P G I E I E S R L G G TG A 12 HLA-DRB1*0301 (DR17) 15-mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5Score 64 A F E I E I N G Q L V F S K L 26 39 Y L E L A S A V K E Q Y P GI 25 72 Q L V F S K L E N G G F P Y E 23 62 T G A F E I E I N G Q L V FS 22 24 R I V V E Y C E P C G F E A T 19 71 G Q L V F S K L E N G G F PY 19 86 E K D L I E A I R R A S N G E 19  7 Q T S V A P P P E E V E P GS 18 23 V R I V V E Y C E P C G F E A 18 50 Y P G I E I E S R L G G T GA 18 90 I E A I R R A S N G E T L E K 18 20 G S G V R I V V E Y C E P CG 17 87 K D L I E A I R R A S N G E T 17 99 G E T L E K I T N S R P P CV 16 28 E Y C E P C G F E A T Y L E L 15 37 A T Y L E L A S A V K E Q YP 14 48 E Q Y P G I E I E S R L G G T 14 78 L E N G G F P Y E K D L I EA 14 14 P E E V E P G S G V R I V V E 13 70 N G Q L V F S K L E N G G FP 13 43 A S A V K E Q Y P G I E I E S 12 52 G I E I E S R L G G T G A FE 12 54 E I E S R L G G T G A F E I E 12 74 V F S K L E N G G F P Y E KD 12 82 G F P Y E K D L I E A I R R A 12 HLA-DRB1*0401 (DR4Dw4) 15-mersPos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 Score 36 E A T Y L E L A S A V K E Q Y28 62 T G A F E I E I N G Q L V F S 28 86 E K D L I E A I R R A S N G E26 87 K D L I E A I R R A S N G E T 26 90 I E A I R R A S N G E T L E K26 72 Q L V F S K L E N G G F P Y E 22 82 G F P Y E K D L I E A I R R A22 50 Y P G I E I E S R L G G T G A 20 99 G E T L E K I T N S R P P C V20 26 V V E Y C E P C G F E A T Y L 16 32 P C G F E A T Y L E L A S A V16 47 K E Q Y P G I E I E S R L G G 16 80 N G G F P Y E K D L I E A I R16 14 P E E V E P G S G V R I V V E 14 20 G S G V R I V V E Y C E P C G14 22 G V R I V V E Y C E P C G F E 14 37 A T Y L E L A S A V K E Q Y P14 39 Y L E L A S A V K E Q Y P G I 14 56 E S R L G G T G A F E I E I N14 64 A F E I E I N G Q L V F S K L 14 66 E I E I N G Q L V F S K L E N14 10 V A P P P E E V E P G S G V R 12 12 P P P E E V E P G S G V R I V12 16 E V E P G S G V R I V V E Y C 12 29 Y C E P C G F E A T Y L E L A12 30 C E P C G F E A T Y L E L A S 12 31 E P C G F E A T Y L E L A S A12 34 G F E A T Y L E L A S A V K E 12 35 F E A T Y L E L A S A V K E Q12 42 L A S A V K E Q Y P G I E I E 12 48 E Q Y P G I E I E S R L G G T12 49 Q Y P G I E I E S R L G G T G 12 53 I E I E S R L G G T G A F E I12 58 R L G G T G A F E I E I N G Q 12 59 L G G T G A F E I E I N G Q L12 61 G T G A F E I E I N G Q L V F 12 63 G A F E I E I N G Q L V F S K12 67 I E I N G Q L V F S K L E N G 12 68 E I N G Q L V F S K L E N G G12 69 I N G Q L V F S K L E N G G F 12 85 Y E K D L I E A I R R A S N G12 93 I R R A S N G E T L E K I T N 12 94 R R A S N G E T L E K I T N S12 96 A S N G E T L E K I T N S R P 12 97 S N G E T L E K I T N S R P P12

TABLE 2 HLA peptide motif search results (“Start Position” = SEQ ID NO:2 Start Position) HLA peptide motif search results User Parameters andScoring Information method selected to limit number of results explicitnumber number of results requested 20 HLA molecule type selected A1length selected for subsequences to be scored 9 echoing mode selectedfor input sequence Y echoing format numbered lines length of user'sinput peptide sequence 115 number of subsequence scores calculated 107number of top-scoring subsequences reported 20 back in scoring outputtable Scoring Results Score (Estimate of Half Time of Disassociation ofa Molecule Subsequence Containing Start Residue This Rank PositionListing Subsequence 1 77 KLENGGFPY 225.000 2 16 EVEPGSGVR 90.000 3 29YCEPCGFEA 45.000 4 39 YLELASAVK 36.000 5 2 SGEPGQTSV 2.250 6 26VVEYCEPCG 1.800 7 96 ASNGETLEK 1.500 8 101 TLEKITNSR 0.900 9 89LIEAIRRAS 0.900 10 54 EIESRLGGT 0.900 11 66 EIEINGQLV 0.900 12 52GIEIESRLG 0.900 13 86 EKDLIEAIR 0.500 14 42 LASAVKEQY 0.500 15 31EPCGFEATY 0.250 16 69 INGQLVFSK 0.250 17 34 GFEATYLEL 0.225 18 98NGETLEKIT 0.225 19 61 GTGAFEIEI 0.125 20 79 ENGGFPYEK 0.100 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A1 length selected for subsequences to be scored 10 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 66 EIEInGQLVF 45.000 2 16 EVEPgSGVRI18.000 3 29 YCEPcGFEAT 9.000 4 26 VVEYcEPCGF 9.000 5 52 GIEIeSRLGG 4.5006 2 SGEPgQTSVA 2.250 7 89 LIEAiRRASN 1.800 8 20 GSGVrIVVEY 1.500 9 86EKDLiEAIRR 1.250 10 98 NGETlEKITN 1.125 11 95 RASNgETLEK 1.000 12 68EINGqLVFSK 1.000 13 54 EIESrLGGTG 0.900 14 41 ELASaVKEQY 0.500 15 100ETLEkITNSR 0.250 16 46 VQEKyPGIEI 0.225 17 39 YLELaSAVKE 0.180 18 77KLENgGFPYE 0.180 19 76 SKLEnGGFPY 0.125 20 48 EQYPgIEIES 0.075 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A_0201 length selected for subsequences to be scored 9 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 9 SVAPPPEEV 2.982 2 104 KITNSRPPC2.391 3 10 ITNSRPPCV 1.642 4 25 IVVEYCEPC 1.485 5 65 FEIEINGQL 1.018 647 KEQYPGIEI 0.710 7 88 DLIEAIRRA 0.703 8 59 LGGTGAFEI 0.671 9 61GTGAFEIEI 0.551 10 81 GGFPYEKDL 0.516 11 37 ATYLELASA 0.508 12 35FEATYLELA 0.501 13 15 EEVEPGSGV 0.416 14 17 VEPGSGVRI 0.345 15 97SNGETLEKI 0.315 16 70 NGQLVFSKL 0.265 17 22 GVRIVVEYC 0.205 18 45AVKEQYPGI 0.196 19 85 YEKDLIEAI 0.151 20 38 TYLELASAV 0.147 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A_0201 length selected for subsequences to be scored 10 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 58 RLGGtGAFEI 60.510 2 104KITNsRPPCV 33.472 3 65 FEIEiNGQLV 25.506 4 83 FPYEkDLIEA 4.502 5 33CGFEaTYLEL 3.173 6 1 MSGEpGQTSV 3.165 7 37 ATLYeLASAV 3.091 8 50YPGIeIESRL 0.641 9 69 INGQlVFSKL 0.450 10 17 VEPGsGVRIV 0.434 11 24RIVVeYCEPC 0.335 12 53 IEIEsRLGGT 0.302 13 60 GGTGaFEIEI 0.259 14 8TSVApPPEEV 0.222 15 44 SAVKeQYPGI 0.217 16 21 SGVRiVVEYC 0.201 17 55IESRlGGTGA 0.164 18 80 NGGFpYEKDL 0.139 19 81 GGFPyEKDLI 0.123 20 105ITNSrPPCVI 0.101 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected A_0205 length selected for subsequences tobe scored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 65 FEIEINGQL8.820 2 25 IVVEYCEPC 3.060 3 9 SVAPPPEEV 2.000 4 104 KITNSRPPC 1.500 581 GGFPYEKDL 1.260 6 45 AVKEQYPGI 1.200 7 70 NGQLVFSKL 0.700 8 47KEQYPGIEI 0.420 9 105 ITNSRPPCV 0.340 10 37 ATYLELASA 0.300 11 35FEATYLELA 0.252 12 17 VEPGSGVRI 0.238 13 61 GTGAFEIEI 0.200 14 97SNGETLEKI 0.150 15 30 CEPCGFEAT 0.140 16 85 YEKDLIEAI 0.126 17 51PGIEIESRL 0.105 18 59 LGGTGAFEI 0.102 19 22 GVRIVVEYC 0.100 20 15EEVEPGSGV 0.084 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected A_0205 length selected for subsequences tobe scored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 33 CGFEaTYLEL 6.300 2 104 KITNsRPPCV6.000 3 65 FEIEiNGQLV 2.520 4 53 IEIEsRLGGT 1.428 5 83 FPYEkDLIEA 1.3506 58 RLGGtGAFEI 1.200 7 69 INGQlVFSKL 1.190 8 50 YPGIeIESRL 1.050 9 37ATYLeLASAV 0.600 10 1 MSGEpGQTSV 0.510 11 80 NGGFpYEKDL 0.420 12 106TNSRpPCVIL 0.350 13 24 RIVVeYCEPC 0.300 14 44 SAVKeQYPGI 0.200 15 17VEPGsGVRIV 0.190 16 105 ITNSrPPCVI 0.170 17 97 SNGEtLEKIT 0.150 18 55IESRlGGTGA 0.119 19 60 GGTGaFEIEI 0.100 20 92 AIRRaSNGET 0.100 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A24 length selected for subsequences to be scored 9 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 34 GFEATYLEL 33.000 2 49 QYPGIEIES11.550 3 70 NGQLVFSKL 11.088 4 38 TYLELASAV 10.800 5 82 GFPYEKDLI 7.5006 81 GGFPYEKDL 4.800 7 107 NSRPPCVIL 4.800 8 75 FSKLENGGF 2.000 9 97SNGETLEKI 1.320 10 45 AVKEQYPGI 1.200 11 61 GTGAFEIEI 1.100 12 59LGGTGAFEI 1.100 13 65 FEIEINGQL 1.008 14 51 PGIEIESRL 1.008 15 106TNSRPPCVI 1.000 16 84 PYEKDLIEA 0.825 17 94 RRASNGETL 0.800 18 28EYCEPCGFE 0.600 19 32 PCGFEATYL 0.400 20 47 KEQYPGIEI 0.330 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A24 length selected for subsequences to be scored 10 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 64 AFEIeINGQL 42.000 2 74 VFSKlENGGF10.000 3 84 PYEKdLIEAI 9.000 4 69 INGQlVFSKL 7.392 5 28 EYCEpCGFEA 6.6006 50 YPGIeIESRL 5.600 7 33 CGFEaTYLEL 5.280 8 106 TNSRpPCVIL 4.000 9 31EPCGfEATYL 4.000 10 80 NGGFpYEKDL 4.000 11 26 VVEYcEPCGF 3.000 12 66EIEInGQLVF 3.000 13 58 RLGGtGAFEI 2.200 14 56 ESRLgGTGAF 2.000 15 16EVEPgSGVRI 1.800 16 96 ASNGeTLEKI 1.650 17 105 ITNSrPPCVI 1.500 18 44SAVKeQYPGI 1.500 19 81 GGFPyEKDLI 1.200 20 60 GGTGaFEIEI 1.100 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A3 length selected for subsequences to be scored 9 echoing modeselected for input sequence Y echoing format numbered lines length ofuser's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 77 KLENGGFPY 36.000 2 39 YLELASAVK20.000 3 101 TLEKITNSR 6.000 4 61 GTGAFEIEI 0.540 5 69 INGQLVFSK 0.360 696 ASNGETLEK 0.300 7 22 GVRIVVEYC 0.270 8 79 ENGGFPYEK 0.162 9 25IVVEYCEPC 0.135 10 45 AVKEQYPGI 0.090 11 37 ATYLELASA 0.075 12 42LASAVKEQY 0.060 13 104 KITNSRPPC 0.060 14 50 YPGIEIESR 0.060 15 72QLVFSKLEN 0.060 16 16 EVEPGSGVR 0.054 17 31 EPCGFEATY 0.054 18 9SVAPPPEEV 0.045 19 87 KDLIEAIRR 0.036 20 27 VEYCEPCGF 0.030 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A3 length selected for subsequences to be scored 10 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 68 EINGqLVFSK 8.100 2 58 RLGGtGAFEI2.700 3 41 ELASaVQEKY 1.800 4 78 LENGgFPYEK 0.810 5 95 RASNgETLEK 0.4006 20 GSGVrIVVEY 0.270 7 100 ETLEkITNSR 0.203 8 26 VVEYcEPCGF 0.200 9 77KLENgGPPYE 0.180 10 66 EIEInGQLVF 0.120 11 24 RIVVeYCEPC 0.090 12 104KITNsRPPCV 0.060 13 37 ATYLeLASAV 0.050 14 38 TYLElASAVK 0.045 15 83FPYEkDLIEA 0.045 16 105 ITNSrPPCVI 0.045 17 72 QLVFsKLENG 0.045 18 30CEPCgFEATY 0.036 19 22 GVRIvVEYCE 0.027 20 16 EVEPgSGVRI 0.027 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A_1101 length selected for subsequences to be scored 9 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 39 YLELASAVK 0.400 2 69 INGQLVFSK0.120 3 16 EVEPGSGVR 0.120 4 101 TLEKITNSR 0.080 5 61 GTGAFEIEI 0.060 650 YPGIEIESR 0.040 7 96 ASNGETLEK 0.040 8 87 KDLIEAIRR 0.036 9 77LENGGFPY 0.036 10 79 ENGGFPYEK 0.024 11 9 SVAPPPEEV 0.020 12 45AVKEQYPGI 0.020 13 37 ATYLELASA 0.020 14 34 GFEATYLEL 0.012 15 105ITNSRPPCV 0.010 16 22 GVRIVVEYC 0.006 17 38 TYLELASAV 0.006 18 82GFPYEKDLI 0.006 19 29 YCEPCGFEA 0.006 20 73 LVFSKLENG 0.004 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A_3101 length selected for subsequences to be scored 9 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 101 TLEKITNSR 2.000 2 16 EVEPGSGVR0.600 3 50 YPGIEIESR 0.400 4 87 KDLIEAIRR 0.240 5 39 YLELASAVK 0.200 677 KLENGGFPY 0.180 7 37 ATYLELASA 0.060 8 69 INGQLVFSK 0.024 9 45AVKEQYPGI 0.020 10 61 GTGAFEIEI 0.020 11 9 SVAPPPEEV 0.020 12 24RIVVEYCEP 0.012 13 34 GFEATYLEL 0.012 14 73 LVFSKLENG 0.012 15 38TYLELASAV 0.012 16 105 ITNSRPPCV 0.010 17 72 QLVFSKLEN 0.008 18 82GFPYEKDLI 0.006 19 104 KITNSRPPC 0.006 20 79 ENGGFPYEK 0.006 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A_3302 length selected for subsequences to be scored 9 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 16 EVEPGSGVR 45.000 2 101 TLEKITNSR9.000 3 50 YPGIEIESR 3.000 4 66 EIEINGQLV 1.500 5 56 ESRLGGTGA 1.500 654 EIESRLGGT 1.500 7 68 EINGQLVFS 1.500 8 86 EKDLIEAIR 0.900 9 41ELASAVKEQ 0.900 10 88 DLIEAIRRA 0.900 11 96 ASNGETLEK 0.500 12 22GVRIVVEYC 0.500 13 1 MSGEPGQTS 0.500 14 89 LIEAIRRAS 0.500 15 107NSRPPCVIL 0.500 16 9 SVAPPPEEV 0.500 17 38 TYLELASAV 0.500 18 25IVVEYCEPC 0.500 19 45 AVKEQYPGI 0.500 20 49 QYPGIEIES 0.500 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A_3302 length selected for subsequences to be scored 9 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 16 EVEPGSGVR 45.000 2 101 TLEKITNSR9.000 3 50 YPGIEIESR 3.000 4 66 EIEINGQLV 1.500 5 56 ESRLGGTGA 1.500 654 EIESRLGGT 1.500 7 68 EINGQLVFS 1.500 8 86 EKDLIEAIR 0.900 9 41ELASAVKEQ 0.900 10 88 DLIEAIRRA 0.900 11 96 ASNGETLEK 0.500 12 22GVRIVVEYC 0.500 13 1 MSGEPGQTS 0.500 14 89 LIEAIRRAS 0.500 15 107NSRPPCVIL 0.500 16 9 SVAPPPEEV 0.500 17 38 TYLELASAV 0.500 18 25IVVEYCEPC 0.500 19 45 AVKEQYPGI 0.500 20 49 QYPGIEIES 0.500 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A68.1 length selected for subsequences to be scored 9 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 16 EVEPGSGVR 900.000 2 9 SVAPPPEEV12.000 3 50 YPGIEIESR 10.000 4 96 ASNGETLEK 9.000 5 101 TLEKITNSR 5.0006 45 AVKEQYPGI 4.000 7 79 ENGGFPYEK 3.600 8 39 YLELASAVK 3.000 9 61GTGAFEIEI 3.000 10 86 EKDLIEAIR 2.250 11 69 INGQLVFSK 1.200 12 87KDLIEAIRR 1.000 13 105 ITNSRPPCV 1.000 14 37 ATYLELASA 1.000 15 56ESRLGGTGA 0.900 16 25 IVVEYCEPC 0.800 17 73 LVFSKLENG 0.800 18 88DLIEAIRRA 0.600 19 18 EPGSGVRIV 0.600 20 26 VVEYCEPCG 0.600 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A68.1 length selected for subsequences to be scored 9 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 16 EVEPGSGVR 900.000 2 9 SVAPPPEEV12.000 3 50 YPGIEIESR 10.000 4 96 ASNGETLEK 9.000 5 101 TLEKITNSR 5.0006 45 AVKEQYPGI 4.000 7 79 ENGGFPYEK 3.600 8 39 YLELASAVK 3.000 9 61GTGAFEIEI 3.000 10 86 EKDLIEAIR 2.250 11 69 INGQLVFSK 1.200 12 87KDLIEAIRR 1.000 13 105 ITNSRPPCV 1.000 14 37 ATYLELASA 1.000 15 56ESRLGGTGA 0.900 16 25 IVVEYCEPC 0.800 17 73 LVFSKLENG 0.800 18 88DLIEAIRRA 0.600 19 18 EPGSGVRIV 0.600 20 26 VVEYCEPCG 0.600 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected A68.1 length selected for subsequences to be scored 10 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 100 ETLEkITNSR 300.000 2 16EVEPgSGVRI 18.000 3 68 EINGqLVFSK 9.000 4 15 EEVEpGSGVR 9.000 5 95RASNgETLEK 3.000 6 85 YEKDlIEAIR 2.250 7 9 SVAPpPEEVE 1.800 8 86EKDLiEAIRR 1.500 9 73 LVFSkLENGG 1.200 10 25 IVVEyCEPCG 1.200 11 105ITNSrPPCVI 1.000 12 37 ATYLeLASAV 1.000 13 78 LENGgFPYEK 0.900 14 8TSVApPPEEV 0.600 15 22 GVRIvVEYCE 0.600 16 18 EPGSgVRIVV 0.600 17 1MSGEpGQTSV 0.600 18 38 TYLElASAVK 0.600 19 49 QYPGiEIESR 0.500 20 45AVKEqYPGIE 0.400 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B14 length selected for subsequences to bescored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 94 RRASNGETL20.000 2 57 SRLGGTGAF 5.000 3 100 ETLEKITNS 3.375 4 105 ITNSRPPCV 2.0005 88 DLIEAIRRA 1.350 6 18 EPGSGVRIV 1.200 7 70 NGQLVFSKL 1.000 8 81GGFPYEKDL 1.000 9 54 EIESRLGGT 0.900 10 97 SNGETLEKI 0.600 11 91EAIRRASNG 0.450 12 68 EINGQLVFS 0.450 13 65 FEIEINGQL 0.300 14 23VRIVVEYCE 0.300 15 21 SGVRIVVEY 0.300 16 51 PGIEIESRL 0.300 17 104KITNSRPPC 0.250 18 48 EQYPGIEIE 0.225 19 93 IRRANGET 0.200 20 107NSRPPCVIL 0.200 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B14 length selected for subsequences to bescored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 103 EKITnSRPPC6.750 2 33 CGFEaTYLEL 5.000 3 93 IRRAsNGETL 4.000 4 18 EPGSgVRIVV 3.0005 88 DLIEaIRRAS 2.250 6 104 KITNsRPPCV 2.000 7 106 TNSRpPCVIL 1.000 8 50YPGIeIESLR 1.000 9 69 INGQlVFSKL 1.000 10 37 ATYLeLASAV 1.000 11 31EPCGfEATYL 0.900 12 48 EQYPgIEIES 0.750 13 76 SKLEnGGFPY 0.750 14 83FPYEkDLIEA 0.750 15 8 TSVApPPEEV 0.600 16 96 ASNGeTLEKI 0.600 17 44SAVKeQYPGI 0.600 18 57 SRLGgTAGFE 0.500 19 53 IEIEsRLGGT 0.450 20 21SGVRiVVEYC 0.300 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_2705 length selected for subsequences tobe scored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 94 RRASNGETL6000.000 2 57 SRLGGTGAF 1000.000 3 93 IRRASNGET 200.000 4 27 VEYCEPCGF75.000 5 77 KLENGGFPY 45.000 6 39 YLELASAVK 30.000 7 65 FEIEINGQL 30.0008 47 KEQYPGIEI 27.000 9 69 INGQLVFSK 20.000 10 23 VRIVVEYCE 20.000 11101 TLEKITNSR 15.000 12 67 IEINGQLVF 15.000 13 107 NSRPPCVIL 10.000 1496 ASNGETLEK 10.000 15 85 YEKDLIEAI 9.000 16 17 VEPGSGVRI 9.000 17 81GGFPYEKDL 7.500 18 106 TNSRPPCVI 6.000 19 97 SNGETLEKI 6.000 20 75FSKLENGGF 5.000 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_2705 length selected for subsequences tobe scored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 93 IRRAaNGETL2000.000 2 94 RRASnGETLE 60.000 3 78 LENGgFPYEK 30.000 4 95 RASNgETLEK30.000 5 58 RLGGtGAFEI 27.000 6 33 CGFEaTYLEL 25.000 7 106 TNSRpPCVIL20.000 8 71 GQLVfSKLEN 20.000 9 23 VRIVvEYCEP 20.000 10 57 SRLGgTGAFE20.000 11 69 INGQlVFSKL 20.000 12 30 CEPCgFEATY 15.000 13 85 YEKDlIEAIR15.000 14 37 ATYLeLASAV 15.000 15 48 EQYPgIEIES 10.000 16 50 YPGIeIESRL10.000 17 104 KITNsRPPCV 9.000 18 65 FEIEiNGQLV 9.000 19 81 GGFPyEKDLI7.500 20 83 FPYEkDLIEA 5.000 User Parameters and Scoring Informationmethod selected to limit number of results explicit number number ofresults requested 20 HLA molecule type selected B_3501 length selectedfor subsequences to be scored 9 echoing mode selected for input sequenceY echoing format numbered lines length of user's input peptide sequence115 number of subsequence scores calculated 107 number of top-scoringsubsequences reported 20 back in scoring output table Scoring ResultsScore (Estimate of Half Time of Disassociation of a Molecule SubsequenceContaining Start Residue This Rank Position Listing Subsequence 1 31EPCGFEATY 40.000 2 75 FSKLENGGF 22.500 3 107 NSRPPCVIL 15.000 4 42LASAVKEQY 6.000 5 18 EPGSGVRIV 4.000 6 45 AVKEQYPGI 2.400 7 21 SGVRIVVEY2.000 8 56 ESRLGGTGA 1.500 9 77 KLENGGFPY 1.200 10 81 GGFPYEKDL 1.000 111 MSGEPGQTS 1.000 12 70 NGQLVFSKL 1.000 13 97 SNGETLEKI 0.800 14 83FPYEKDLIE 0.400 15 61 GTGAFEIEI 0.400 16 59 LGGTGAFEI 0.400 17 106TNSRPPCVI 0.400 18 50 YPGIEIESR 0.300 19 22 GVRIVVEYC 0.300 20 11APPPEEVEP 0.300 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_3501 length selected for subsequences tobe scored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 31 EPCGfEATYL30.000 2 50 YPGIeIESRL 20.000 3 56 ESRLgGTGAF 15.000 4 20 GSGVrIVVEY10.000 5 83 FPYEkDLIEA 6.000 6 18 EPGSgVRIVV 4.000 7 33 CGFEaTYLEL 2.0008 1 MSGEpGQTSV 2.000 9 96 ASNGeTLEKI 2.000 10 41 ELASaVKEQY 2.000 11 44SAVKeQYPGI 1.200 12 69 INGQlVFSKL 1.000 13 8 TSVApPPEEV 1.000 14 80NGGFpYEKDL 1.000 15 106 TNSRpPCVIL 1.000 16 58 RLGGtGAFEI 0.800 17 81GGFPyEKDLI 0.600 18 26 VVEYcEPCGF 0.450 19 36 EATYlELASA 0.450 20 12PPPEaVEPGS 0.400 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_3901 length selected for subsequences tobe scored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 94 RRASNGETL15.000 2 34 GFEATYLEL 9.000 3 38 TYLELASAV 4.000 4 66 EIEINGQLV 3.000 52 SGEPGQTSV 3.000 6 97 SNGETLEKI 3.000 7 70 NGQLVFSKL 3.000 8 81GGFPYEKDL 3.000 9 18 EPGSGVRIV 1.500 10 65 FEIEINGQL 1.200 11 57SRLGGTGAF 1.000 12 106 TNSRPPCVI 1.000 13 9 SVAPPPEEV 1.000 14 59LGGTGAFEI 1.000 15 105 ITNSRPPCV 1.000 16 107 NSRPPCVIL 0.900 17 45AVKEQYPGI 0.600 18 51 PGIEIESRL 0.600 19 88 DLIEAIRRA 0.600 20 100ETLEKITNS 0.600 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_3901 length selected for subsequences tobe scored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 33 CGFEaTYLEL12.000 2 64 AFEIeINGQL 9.000 3 93 IRRAsNGETL 4.500 4 46 VKEQyPGIEI 3.0005 16 EVEPgSGVRI 3.000 6 106 TNSRpPCVIL 3.000 7 69 INGQlVFSKL 3.000 8 31EPCGfEATYL 3.000 9 44 SAVKeQYPGI 2.000 10 1 MSGEpGQTSV 2.000 11 8TSVApPPEEV 2.000 12 37 ATYLeLASAV 2.000 13 80 NGGFpYEKDL 1.500 14 50YPGIeIESRL 1.500 15 96 ASNGeTLEKI 1.500 16 58 RLGGtGAFEI 1.000 17 105ITNSrPPCVI 1.000 18 81 GGFPyEKDLI 1.000 19 104 KITNsRPPCV 1.000 20 83FPYEkDLIEA 0.600 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B40 length selected for subsequences to bescored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 65 FEIEINGQL80.000 2 3 GEPGQTSVA 40.000 3 35 FEATYLELA 40.000 4 15 EEVEPGSGV 24.0005 67 IEINGQLVF 16.000 6 81 GGFPYEKDL 8.000 7 27 VEYCEPCGF 8.000 8 47KEQYPGIEI 6.000 9 17 VEPGSGVRI 4.000 10 30 CEPCGFEAT 4.000 11 99GETLEKITN 2.400 12 90 IEAIRRASN 2.400 13 37 ATYLELASA 2.000 14 85YEKDLIEAI 2.000 15 53 IEIESRLGG 1.600 16 40 LELASAVKE 0.800 17 107NSRPPCVIL 0.750 18 29 YCEPCGFEA 0.500 19 70 NGQLVFSKL 0.500 20 78LENGGFPYE 0.400 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B40 length selected for subsequences to bescored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 55 IESRlGGTGA20.000 2 53 IEIEIsRLGGT 16.000 3 65 FEIEiNGQLV 16.000 4 67 IEINgQLVFS16.000 5 99 GETLeKITNS 8.000 6 35 FEATyLELAS 8.000 7 87 KDLIeAIRRA 5.0008 17 VEPGsGVRIV 4.000 9 30 CEPCgFEATY 4.000 10 33 CGFEaTYLEL 2.000 11 15EEVEpGSGVR 1.600 12 81 GGFPyEKDLI 1.600 13 27 VEYCePCGFE 1.200 14 83FPYEkDLIEA 1.000 15 40 LELAsAVKEQ 0.800 16 3 GEPGqTSVAP 0.800 17 90IEAIrRASNG 0.800 18 106 TNSRpPCVIL 0.750 19 8 TSVApPPEEV 0.600 20 2SGEPgQTSVA 0.500 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_5201 length selected for subsequences tobe scored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 18 EPGSGVRIV75.000 2 67 IEINGQLVF 22.500 3 59 LGGTGAFEI 11.250 4 98 NGETLEKIT 11.0005 19 PGSGVRIVV 10.000 6 106 TNSRPPCVI 10.000 7 48 EQYPGIEIE 9.900 8 2SGEPGQTSV 9.000 9 81 GGFPYEKDL 6.600 10 38 TYLELASAV 4.800 11 27VEYCEPCGF 3.750 12 83 FPYEKDLIE 3.000 13 17 VEPGSGVRI 3.000 14 70NGQLVFSKL 2.400 15 85 YEKDLIEAI 2.200 16 3 GEPGQTSVA 2.200 17 82GFPYEKDLI 2.200 18 97 SNGETLEKI 2.178 19 61 GTGAFEIEI 1.800 20 105ITNSRPPCV 1.500 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_5201 length selected for subsequences tobe scored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 18 EPGSgVRIVV100.000 2 17 VEPGsGVRIV 45.000 3 81 GGFPyEKDLI 33.000 4 105 ITNSrPPCVI15.000 5 37 ATYLeLASAV 12.000 6 66 EIEInGQLVF 9.000 7 33 CGFEaTYLEL9.000 8 60 GGTGaFEIEI 7.500 9 2 SGEPgQTSVA 6.600 10 83 FPYEkDLIEA 3.30011 1 MSGEpGQTSV 2.700 12 97 SNGEtLEKIT 2.640 13 65 FEIEiNGQLV 2.640 1450 YPGIeIESRL 2.400 15 48 EQYPgIEIES 2.400 16 106 TNSRpPCVIL 2.000 17 96ASNGeTLEKI 1.815 18 58 RLGGtGAFEI 1.500 19 8 TSVApPPEEV 1.320 20 59LGGTgAFEIE 1.238 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B60 length selected for subsequences to bescored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 65 FEIEINGQL387.200 2 17 VEPGSGVRI 17.600 3 15 EEVEPGSGV 16.000 4 47 KEQYPGIEI16.000 5 85 YEKDLIEAI 8.800 6 107 NSRPPCVIL 8.000 7 35 FEATYLELA 8.000 870 NGQLVFSKL 4.840 9 3 GEPGQTSVA 4.000 10 81 GGFPYEKDL 4.000 11 30CEPCGFEAT 4.000 12 67 IEINGQLVF 3.200 13 90 IEAIRRASN 2.400 14 99GETLEKITN 2.400 15 40 LELASAVKE 1.760 16 53 IEIESRLGG 1.600 17 51PGIEIESRL 0.968 18 55 IESRLGGTG 0.880 19 34 GFEATYLEL 0.800 20 94RRASNGETL 0.800 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B60 length selected for subsequences to bescored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 65 FEIEiNGQLV16.000 2 106 TNSRpPCVIL 16.000 3 53 IEIEsRLGGT 8.000 4 33 CGFEaTYLEL8.000 5 17 VEPGsGVRIV 8.000 6 55 IESRlGGTGA 8.000 7 69 INGQlVFSKL 4.8408 50 YPGIeIESRL 4.840 9 80 NGGFpYEKDL 4.000 10 31 EPCGfEATYL 4.000 11 35FEATyLELAS 3.520 12 67 IEINgQLVFS 3.200 13 87 KDLIeAIRRA 1.100 14 78LENGgFPYEK 0.800 15 15 EEVEpGSGVR 0.800 16 99 GETLeKITNS 0.800 17 30CEPCgFEATY 0.800 18 90 IEAIrRASNG 0.800 19 3 GEPGqTSVAP 0.800 20 40LELAsAVKEQ 0.800 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B61 length selected for subsequences to bescored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 15 EEVEPGSGV80.000 2 35 FEATYLELA 40.000 3 3 GEPGQTSVA 22.000 4 65 FEIEINGQL 16.0005 85 YEKDLIEAI 16.000 6 17 VEPGSGVRI 8.000 7 47 KEQYPGIEI 8.000 8 30CEPCGFEAT 4.000 9 99 GETLEKITN 2.640 10 90 IEAIRRASN 2.400 11 27VEYCEPCGF 1.600 12 67 IEINGQLVF 1.600 13 2 SGEPGQTSV 1.000 14 18EPGSGVRIV 1.000 15 105 ITNSRPPCV 1.000 16 37 ATYLELASA 1.000 17 53IEIESRLGG 0.800 18 40 LELASAVKE 0.800 19 81 GGFPYEKDL 0.660 20 29YCEPCGFEA 0.500 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B61 length selected for subsequences to bescored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 65 FEIEiNGQLV80.000 2 17 VEPGsGVRIV 40.000 3 55 IESRlGGTGA 20.000 4 87 KDLIeAIRRA10.000 5 53 IEIEsRLGGT 8.000 6 14 PEEVePGSGV 4.000 7 99 GETLeKITNS 3.5208 37 ATYLeLASAV 2.000 9 8 TSVApPPEEV 2.000 10 67 IEINgQLVFS 1.600 11 35FEATyLELAS 1.600 12 1 MSGEpGQTSV 1.000 13 18 EPGSgVRIVV 1.000 14 36EATYlELASA 1.000 15 83 FPYEkDLIEA 1.000 16 15 EEVEpGSGVR 0.800 17 27VEYCePCGFE 0.800 18 30 CEPCgFEATY 0.800 19 90 IEAIrRASNG 0.800 20 40LELAaAVKEQ 0.800 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B62 length selected for subsequences to bescored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 77 KLENGGFPY24.000 2 21 SGVRIVVEY 4.800 3 75 FSKLENGGF 3.000 4 31 EPCGFEATY 2.640 588 DLIEAIRRA 2.200 6 42 LASAVKEQY 2.000 7 48 EQYPGIEIE 0.960 8 71GQLVFSKLE 0.800 9 6 GQTSVAPPP 0.800 10 67 IEINGQLVF 0.686 11 22GVRIVVEYC 0.660 12 58 RLGGTGAFE 0.480 13 57 SRLGGTGAF 0.480 14 18EPGSGVRIV 0.400 15 59 LGGTGAFEI 0.400 16 56 ESRLGGTGA 0.360 17 45AVKEQYPGI 0.330 18 104 KITNSRPPC 0.250 19 72 QLVFSKLEN 0.240 20 61GTGAFEIEI 0.240 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B62 length selected for subsequences to bescored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 41 ELASaVKEQY40.000 2 58 RLGGtGAFEI 9.600 3 66 EIEInGQLVF 7.920 4 56 ESRLgGTGAF 6.0005 20 GSGVrIVVEY 4.800 6 92 AIRRaSNGET 1.500 7 48 EQYPgIEIES 1.152 8 26VVEYcEPCGF 0.600 9 24 RIVVeYCEPC 0.500 10 104 KITNsRPPCV 0.500 11 71GQLVfSKLEN 0.480 12 76 SKLEnGGFPY 0.440 13 88 DLIEaIRRAS 0.440 14 6GQTSvSPPPE 0.400 15 1 MSGEpGQTSV 0.264 16 18 EPGSgVRIVV 0.264 17 69INGQlVFSKL 0.260 18 21 SGVRiVVEYC 0.220 19 30 CEPCgFEATY 0.220 20 74VFSKlENGGF 0.200 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B7 length selected for subsequences to bescored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 107 NSRPPCVIL60.000 2 45 AVKEQYPGI 6.000 3 22 GVRIVVEYC 5.000 4 70 NGQLVFSKL 4.000 581 GGFPYEKDL 4.000 6 18 EPGSGVRIV 4.000 7 9 SVAPPPEEV 1.500 8 56ESRLGGTGA 1.000 9 106 TNSRPPCVI 0.600 10 11 APPPEEVEP 0.600 11 25IVVEYCEPC 0.500 12 65 FEIEINGQL 0.400 13 61 GTGAFEIEI 0.400 14 31EPCGFEATY 0.400 15 94 RRASNGETL 0.400 16 59 LGGTGAFEI 0.400 17 51PGIEIESRL 0.400 18 32 PCGFEATYL 0.400 19 97 SNGETLEKI 0.400 20 92AIRRASNGE 0.300 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B7 length selected for subsequences to bescored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 50 YPGIeIESRL80.000 2 31 EPCGfEATYL 80.000 3 18 EPGSgVRIVV 6.000 4 106 TNSRpPCVIL6.000 5 80 NGGFpYEKDL 4.000 6 69 INGQlVFSKL 4.000 7 93 IRRAsNGETL 4.0008 33 CGFEaTYLEL 4.000 9 92 AIRRaSNGET 3.000 10 83 FPYEkDLIEA 2.000 11 44SAVKeQYPGI 1.200 12 96 ASNGeTLEKI 1.200 13 11 APPPeEVEPG 0.600 14 16EVEPgSGVRI 0.600 15 37 ATYLeLASAV 0.600 16 105 ITNSrPPCVI 0.600 17 22GVRIvVEYCE 0.500 18 60 GGTGaFEIEI 0.400 19 81 GGFPyEKDLI 0.400 20 58RLGGtGAFEI 0.400 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B8 length selected for subsequences to bescored 8 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 108 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 83 FPYEKDLI 6.0002 107 NSRPPCVI 1.000 3 91 EAIRRASN 0.800 4 20 GSGVRIVV 0.600 5 18EPGSGVRI 0.400 6 95 RASNGETL 0.400 7 100 ETLEKITN 0.300 8 105 ITNSRPPC0.200 9 10 VAPPPEEV 0.120 10 73 LVFSKLEN 0.100 11 43 ASAVKEQY 0.100 1222 GVRIVVEY 0.100 13 36 EATYLELA 0.080 14 31 EPCGFEAT 0.080 15 66EIEINGQL 0.080 16 4 EPGQTSVA 0.080 17 33 CGFEATYL 0.060 18 71 GQLVFSKL0.060 19 56 ESRLGGTG 0.040 20 106 TNSRPPCV 0.030 User Parameters andScoring Information method selected to limit number of results explicitnumber number of results requested 20 HLA molecule type selected B8length selected for subsequences to be scored 8 echoing mode selectedfor input sequence Y echoing format numbered lines length of user'sinput peptide sequence 115 number of subsequence scores calculated 108number of top-scoring subsequences reported 20 back in scoring outputtable Scoring Results Score (Estimate of Half Time of Disassociation ofa Molecule Subsequence Containing Start Residue This Rank PositionListing Subsequence 1 83 FPYEKDLI 6.000 2 107 NSRPPCVI 1.000 3 91EAIRRASN 0.800 4 20 GSGVRIVV 0.600 5 18 EPGSGVRI 0.400 6 95 RASNGETL0.400 7 100 ETLEKITN 0.300 8 105 ITNSRPPC 0.200 9 10 VAPPPEEV 0.120 1073 LVFSKLEN 0.100 11 43 ASAVKEQY 0.100 12 22 GVRIVVEY 0.100 13 36EATYLELA 0.080 14 31 EPCGFEAT 0.080 15 66 EIEINGQL 0.080 16 4 EPGQTSVA0.080 17 33 CGFEATYL 0.060 18 71 GQLVFSKL 0.060 19 56 ESRLGGTG 0.040 20106 TNSRPPCV 0.030 User Parameters and Scoring Information methodselected to limit number of results explicit number number of resultsrequested 20 HLA molecule type selected Cw_0702 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence115 number of subsequence scores calculated 106 number of top-scoringsubsequences reported 20 back in scoring output table Scoring ResultsScore (Estimate of Half Time of Disassociation of a Molecule SubsequenceContaining Start Residue This Rank Position Listing Subsequence 1 20GSGVrIVVEY 38.400 2 30 CEPCgFEATY 16.000 3 41 ELASaVKEQY 16.000 4 50YPGIeIESRL 7.920 5 76 SKLEnGGFPY 4.000 6 69 INGQlVFSKL 2.880 7 18EPGSgVRIVV 2.400 8 33 CGFEaTYLEL 1.440 9 80 HGGFpYEKDL 1.440 10 56ESRLgGTGAF 1.200 11 93 IRRAsNGETL 1.200 12 64 AFEIeINGQL 1.200 13 66EIEInGQLVF 1.000 14 35 FEATyLELAS 0.960 15 87 KDLIeAIRRA 0.800 16 97SNGEtLEKIT 0.800 17 17 VEPGsGVRIV 0.800 18 21 SGVRiVVEYC 0.800 19 28EYCEpCGFEA 0.720 20 48 EQYPgIEIES 0.672 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B8 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 50 YPGIeIESRL 0.800 2 93 IRRAsNGETL 0.400 3 31 EPCGfEATYL0.320 4 104 KITNsRPPCV 0.300 5 18 EPGSgVRIVV 0.240 6 56 ESRLgGTGAF 0.2007 44 SAVKeQYPGI 0.200 8 92 AIRRaSNGET 0.200 9 69 INGQlVFSKL 0.200 10 106TNSRpPCVIL 0.200 11 42 LASAvKEQYP 0.160 12 33 CGFEaTYLEL 0.060 13 105ITNSrPPCVI 0.050 14 58 RLGGtGAFEI 0.050 15 96 ASNGeTLEKI 0.050 16 1MSGEpGQTSV 0.045 17 75 FSKLeNGGFP 0.040 18 80 NGGFpYEKDL 0.040 19 72QLVFsKLENG 0.040 20 53 IEIEsRLGGT 0.030 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B_2702 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format numbered lines length of user's input peptidesequence 115 number of subsequence scores calculated 107 number oftop-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 57 SRLGGTGAF 200.000 2 94 RRASNGETL 180.000 3 93 IRRASNGET20.000 4 27 VEYCEPCGF 15.000 5 77 KLENGGFPY 9.000 6 67 IEINGQLVF 3.000 747 KEQYPGIEI 2.700 8 23 VRIVVEYCE 2.000 9 42 LASAVKEQY 1.000 10 75FSKLENGGF 1.000 11 85 YEKDLIEAI 0.900 12 17 VEPGSGVRI 0.900 13 65FEIEINGQL 0.900 14 97 SNGETLEKI 0.600 15 106 TNSRPPCVI 0.600 16 37ATYLELASA 0.500 17 21 SGVRIVVEY 0.500 18 107 NSRPPCVIL 0.300 19 30CEPCGFEAT 0.300 20 48 EQYPGIEIE 0.300 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B_2702 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 93 IRRAsNGETL 60.000 2 94 RRASnGETLE 6.000 3 30 CEPCgFEATY3.000 4 58 RLGGtGAFEI 2.700 5 23 VRIVvEYCEP 2.000 6 57 SRLGgTGAFE 2.0007 48 EQYPgIEIES 1.500 8 26 VVEYcEPCGF 1.000 9 20 GSGVrIVVEY 1.000 10 71GQLVfSKLEN 1.000 11 41 ELASaVKEQY 0.900 12 33 CGFEaTYLEL 0.750 13 81GGFPyEKDLI 0.750 14 106 TNSRpPCVIL 0.600 15 69 INGQlVFSKL 0.600 16 83FPYEkDLIEA 0.500 17 37 ATYLeLASAV 0.500 18 55 IESRlGGTGA 0.300 19 96ASNGeTLEKI 0.300 20 56 ESRLgGTGAF 0.300 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B_3701 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 65 FEIEiNGQLV 10.000 2 67 IEINgQLVFS 5.000 3 81 GGFPyEKDLI5.000 4 87 KDLIeAIRRA 4.000 5 30 CEPCgFEATY 2.000 6 17 VEPGsGVRIV 2.0007 50 YPGIeIESRL 1.500 8 64 AFEIeINGQL 1.500 9 69 INGQlVFSKL 1.500 10 99GETLeKITNS 1.000 11 60 GGTGaFEIEI 1.000 12 46 VKEQyPGIEI 1.000 13 53IEIEsRLGGT 1.000 14 16 EVEPgSGVRI 1.000 15 44 SAVKeQYPGI 1.000 16 105ITNSrPPCVI 1.000 17 96 ASNGeTLEKI 1.000 18 80 NGGFpYEKDL 1.000 19 55IESRlGGTGA 1.000 20 31 EPCGfEATYL 1.000 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B_3801 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format numbered lines length of user's input peptidesequence 115 number of subsequence scores calculated 107 number oftop-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 34 GFEATYLEL 6.000 2 70 NGQLVFSKL 1.560 3 38 TYLELASAV1.040 4 81 GGFPYEKDL 1.000 5 97 SNGETLEKI 0.720 6 66 EIEINGQLV 0.600 7 2SGEPGQTSV 0.600 8 82 GFPYEKDLI 0.600 9 49 QYPGIEIES 0.520 10 18EPGSGVRIV 0.400 11 31 EPCGFEATY 0.400 12 89 LIEAIRRAS 0.390 13 98NGETLEKIT 0.390 14 77 KLENGGFPY 0.300 15 61 GTGAFEIEI 0.300 16 107NSRPPCVIL 0.300 17 75 FSKLENGGF 0.300 18 106 TNSRPPCVI 0.300 19 29YCEPCGFEA 0.300 20 54 EIESRLGGT 0.300 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B_3801 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 64 AFEIeINGQL 7.800 2 31 EPCGfEATYL 4.800 3 66 EIEInGQLVF3.000 4 26 VVEYcEPCGF 3.000 5 50 YPGIeIESRL 2.600 6 74 VFSKlENGGF 2.0007 33 CGFEaTYLEL 2.000 8 69 INGQlVFSKL 1.560 9 106 TNSRpPCVIL 1.000 10 80NGGFpYEKDL 1.000 11 16 EVEPgSGVRI 0.900 12 96 ASNGeTLEKI 0.720 13 34GFEAtYLELA 0.600 14 60 GGTGaFEIEI 0.600 15 58 RLGGtGAFEI 0.600 16 18EPGSgVRIVV 0.520 17 83 FPYEkDLIEA 0.400 18 28 EYCEpCGFEA 0.400 19 1MSGEpGQTSV 0.400 20 2 SGEPgQTSVA 0.300 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B_3902 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format numbered lines length of user's input peptidesequence 115 number of subsequence scores calculated 107 number oftop-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 70 NGQLVFSKL 2.400 2 81 GGFPYEKDL 2.400 3 94 RRASNGETL2.000 4 34 GFEATYLEL 2.000 5 107 NSRPPCVIL 0.600 6 57 SRLGGTGAF 0.500 765 FEIEINGQL 0.480 8 51 PGIEIESRL 0.240 9 32 PCGFEATYL 0.200 10 75FSKLENGGF 0.150 11 86 EKDLIEAIR 0.120 12 6 GQTSVAPPP 0.120 13 71GQLVFSKLE 0.120 14 46 VKEQYPGIE 0.120 15 89 LIEAIRRAS 0.120 16 21SGVRIVVEY 0.120 17 98 NGETLEKIT 0.120 18 36 EATYLELAS 0.120 19 38TYLELASAV 0.120 20 31 EPCGFEATY 0.120 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B_3902 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format numbered lines length of user's input peptidesequence 115 number of subsequence scores calculated 107 number oftop-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 70 NGQLVFSKL 2.400 2 81 GGFPYEKDL 2.400 3 94 RRASNGETL2.000 4 34 GFEATYLEL 2.000 5 107 NSRPPCVIL 0.600 6 57 SRLGGTGAF 0.500 765 FEIEINGQL 0.480 8 51 PGIEIESRL 0.240 9 32 PCGFEATYL 0.200 10 75FSKLENGGF 0.150 11 86 EKDLIEAIR 0.120 12 6 GQTSVAPPP 0.120 13 71GQLVFSKLE 0.120 14 46 VKEQYPGIE 0.120 15 89 LIEAIRRAS 0.120 16 21SGVRIVVEY 0.120 17 98 NGETLEKIT 0.120 18 36 EATYLELAS 0.120 19 38TYLELASAV 0.120 20 31 EPCGFEATY 0.120 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B_3902 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 69 INGQlVFSKL 2.400 2 64 AFEIeINGQL 2.400 3 50 YPGIeIESRL2.400 4 80 NGGFpYEKDL 2.400 5 106 TNSRpPCVIL 2.000 6 31 EPCGfEATYL 2.0007 33 CGFEaTYLEL 2.000 8 48 EQYPgIEIES 1.200 9 76 SKLEnGGFPY 1.000 10 71GQLVfSKLEN 1.000 11 46 VKEQyPGIEI 1.000 12 103 EKITnSRPPC 1.000 13 93IRRAsNGETL 0.600 14 66 EIEInGQLVF 0.500 15 26 VVEYcEPCGF 0.500 16 74VFSKlENGGF 0.500 17 56 ESRLgTGAF 0.150 18 24 RIVVeYCEPC 0.120 19 34GFEAtYLELA 0.120 20 60 GGTGaFEIEI 0.120 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B_4403 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format numbered lines length of user's input peptidesequence 115 number of subsequence scores calculated 107 number oftop-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 67 IEINGQLVF 200.000 2 27 VEYCEPCGF 40.000 3 21 SGVRIVVEY36.000 4 65 FEIEINGQL 20.000 5 35 FEATYLELA 12.000 6 3 GEPGQTSVA 9.000 715 EEVEPGSGV 8.000 8 17 VEPGSGVRI 6.000 9 42 LASAVKEQY 4.500 10 31EPCGFEATY 4.500 11 85 YEKDLIEAI 4.000 12 30 CEPCGFEAT 4.000 13 47KEQYPGIEI 4.000 14 90 IEAIRRASN 3.600 15 53 IEIESRLGG 2.000 16 40LELASAVKE 1.800 17 99 GETLEKITN 1.200 18 75 FSKLENGGF 1.000 19 57SRLGGTGAF 0.900 20 78 LENGGFPYE 0.600 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 20 HLA molecule type selected B_4403 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 30 CEPCgFEATY 120.000 2 53 IEIEsRLGGT 30.000 3 67IEINgQLVFS 30.000 4 65 FEIEiNGQLV 20.000 5 17 VEPGsGVRIV 18.000 6 20GSGVrIVVEY 9.000 7 99 GETLeKITNS 9.000 8 35 FEATyLELAS 8.000 9 55IESRlGGTGA 6.000 10 40 LELAsAVKEQ 5.400 11 87 KDLIeAIRRA 2.250 12 76SKLEnGGFPY 1.800 13 90 IEAIrRASNG 1.800 14 21 SGVRiVVEYC 1.800 15 56ESRLgGTGAF 1.500 16 41 ELASaVKEQY 0.900 17 15 EEVEpGSGVR 0.800 18 96ASNGeTLEKI 0.675 19 3 GEPGqTSVAP 0.600 20 78 LENGgFPYEK 0.600 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected B_5101 length selected for subsequences to be scored 9 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 18 EPGSGVRIV 484.000 2 59 LGGTGAFEI114.400 3 2 SGEPGQTSV 48.400 4 81 GGFPYEKDL 44.000 5 70 NGQLVFSKL 22.0006 31 EPCGFEATY 7.260 7 97 SNGETLEKI 5.856 8 36 EATYLELAS 5.000 9 19PGSGVRIVV 4.840 10 66 EIEINGQLV 4.840 11 45 AVKEQYPGI 4.400 12 82GFPYEKDLI 4.400 13 61 GTGAFEIEI 4.000 14 106 TNSRPPCVI 4.000 15 83FPYEKDLIE 2.860 16 105 ITNSRPPCV 2.600 17 42 LASAVKEQY 2.595 18 51PGIEIESRL 2.420 19 4 EPGQTSVAP 2.200 20 9 SVAPPPEEV 2.200 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected B_5101 length selected for subsequences to be scored 10 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 18 EPGSgVRIVV 440.000 2 44SAVKeQYPGI 220.000 3 31 EPCGfEATYL 220.000 4 81 GGFPyEKDLI 176.000 5 50YPGIeIESRL 157.300 6 60 GGTGaFEIEI 88.000 7 33 CGFEaTYLEL 48.400 8 83FPYEkDLIEA 31.460 9 80 NGGFpYEKDL 22.000 10 36 EATYlELASA 11.000 11 16EVEPgSGVRI 8.800 12 96 ASNGeTLEKI 5.856 13 105 ITNSrPPCVI 5.200 14 37ATYLeLASAV 4.000 15 1 MSGEpGQTSV 3.461 16 21 SGVRiVVEYC 2.420 17 58RLGGtGAFEI 2.420 18 4 EPGQtSVAPP 2.200 19 8 TSVApPPEEV 2.200 20 2SGEPgQTSVA 2.200 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_5102 length selected for subsequences tobe scored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 18 EPGSGVRIV242.000 2 81 GGFPYEKDL 110.000 3 59 LGGTGAFEI 96.800 4 70 NGQLVFSKL48.400 5 2 SGEPGQTSV 24.200 6 51 PGIEIESRL 13.200 7 83 FPYEKDLIE 11.0008 97 SNGETLEKI 10.648 9 38 TYLELASAV 6.600 10 19 PGSGVRIVV 4.840 11 106TNSRPPCVI 4.400 12 61 GTGAFEIEI 4.000 13 82 GFPYEKDLI 4.000 14 31EPCGFEATY 3.630 15 63 GAFEIEING 2.750 16 36 EATYLELAS 2.500 17 50YPGIEIESR 2.420 18 45 AVKEQYPGI 2.420 19 9 SVAPPPEEV 2.200 20 105ITNSRPPCV 2.000 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_5102 length selected for subsequences tobe scored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 44 SAVKeQYPGI726.000 2 50 YPGIeIESRL 400.000 3 81 GGFPyEKDLI 400.000 4 18 EPGSgVRIVV220.000 5 31 EPCGfEATYL 121.000 6 33 CGFEaTYLEL 121.000 7 83 FPYEkDLIEA110.000 8 60 GGTGaFEIEI 88.000 9 80 NGGFpYEKDL 22.000 10 37 ATYLeLASAV11.000 11 96 ASNGeTLEKI 10.648 12 21 SGVRiVVEYC 8.785 13 8 TSVApPPEEV6.600 14 36 EATYlELASA 5.000 15 58 RLGGtGAFEI 4.840 16 16 EVEPgSGVRI4.000 17 105 ITNSrPPCVI 4.000 18 65 FEIEiNGQLV 3.194 19 63 GAFEiEINGQ3.025 20 1 MSGEpGQTSV 2.662 User Parameters and Scoring Informationmethod selected to limit number of results explicit number number ofresults requested 20 HLA molecule type selected B_5103 length selectedfor subsequences to be scored 10 echoing mode selected for inputsequence Y echoing format numbered lines length of user's input peptidesequence 115 number of subsequence scores calculated 106 number oftop-scoring subsequences reported 20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Subsequence Containing Start Residue This Rank Position ListingSubsequence 1 44 SAVKeQYPGI 110.000 2 81 GGFPyEKDLI 52.800 3 18EPGSgVRIVV 44.000 4 60 GGTGaFEIEI 44.000 5 33 CGFEaTYLEL 7.920 6 37ATYLeLASAV 6.600 7 31 EPCGfEATYL 6.600 8 83 FPYEkDLIEA 6.600 9 80NGGFpYEKDL 6.000 10 50 YPGIeIESRL 6.000 11 36 EATYlELASA 5.000 12 21SGVRiVVEYC 2.420 13 2 SGEPgQTSVA 2.420 14 1 MSGEpGQTSV 2.420 15 104KITNsRPPCV 2.420 16 58 RLGGtGAFEI 2.420 17 96 ASNGeTLEKI 2.200 18 8TSVApPPEEV 2.200 19 16 EVEPgSGVRI 2.200 20 105 ITNSrPPCVI 2.000 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested 20 HLA molecule typeselected B_5103 length selected for subsequences to be scored 10 echoingmode selected for input sequence Y echoing format numbered lines lengthof user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported 20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Subsequence Containing Start Residue ThisRank Position Listing Subsequence 1 44 SAVKeQYPGI 110.000 2 81GGFPyEKDLI 52.800 3 18 EPGSgVRIVV 44.000 4 60 GGTGaFEIEI 44.000 5 33CGFEaTYLEL 7.920 6 37 ATYLeLASAV 6.600 7 31 EPCGfEATYL 6.600 8 83FPYEkDLIEA 6.600 9 80 NGGFpYEKDL 6.000 10 50 YPGIeIESRL 6.000 11 36EATYlELASA 5.000 12 21 SGVRiVVEYC 2.420 13 2 SGEPgQTSVA 2.420 14 1MSGEpGQTSV 2.420 15 104 KITNsRPPCV 2.420 16 58 RLGGtGAFEI 2.420 17 96ASNGeTLEKI 2.200 18 8 TSVApPPEEV 2.200 19 16 EVEPgSGVRI 2.200 20 105ITNSrPPCVI 2.000 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_5801 length selected for subsequences tobe scored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 75 FSKLENGGF40.000 2 42 LASAVKEQY 4.500 3 107 NSRPPCVIL 4.000 4 61 GTGAFEIEI 3.000 5105 ITNSRPPCV 3.000 6 37 ATYLELASA 2.400 7 1 MSGEPGQTS 0.880 8 67IEINGQLVF 0.660 9 56 ESRLGGTGA 0.600 10 21 SGVRIVVEY 0.540 11 27VEYCEPCGF 0.400 12 63 GAFEIEING 0.330 13 100 ETLEKITNS 0.317 14 95RASNGETLE 0.300 15 20 GSGVRIVVE 0.240 16 96 ASNGETLEK 0.220 17 44SAVKEQYPG 0.220 18 2 SGEPGQTSV 0.200 19 10 VAPPPEEVE 0.200 20 57SRLGGTGAF 0.200 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected B_5801 length selected for subsequences tobe scored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 56 ESRLgGTGAF12.000 2 20 GSGVrIVVEY 10.800 3 1 MSGEpGQTSV 4.000 4 105 ITNSrPPCVI3.000 5 37 ATYLeLASAV 3.000 6 96 ASNGeTLEKI 2.640 7 44 SAVKeQYPGI 2.0008 8 TSVApPPEEV 2.000 9 74 VFSKlENGGF 0.800 10 61 GTGAfEIEIN 0.480 11 26VVEYcEPCGF 0.400 12 36 EATYlELASA 0.360 13 95 RASNgETLEK 0.330 14 63GAFEiEINGQ 0.264 15 83 FPYEkDLIEA 0.240 16 29 YCEPcGFEAT 0.240 17 33CGFEaTYLEL 0.220 18 43 ASAVkEQYPG 0.220 19 75 FSKLeNGGFP 0.200 20 7QTSVaPPPEE 0.200 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected Cw_0301 length selected for subsequencesto be scored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 65 FEIEINGQL30.000 2 81 GGFPYEKDL 18.000 3 70 NGQLVFSKL 12.000 4 57 SRLGGTGAF 10.0005 34 GFEATYLEL 10.000 6 94 RRASNGETL 5.760 7 27 VEYCEPCGF 5.000 8 67IEINGQLVF 5.000 9 107 NSRPPCVIL 2.000 10 51 PGIEIESRL 1.800 11 15EEVEPGSGV 1.800 12 38 TYLELASAV 1.800 13 21 SGVRIVVEY 1.500 14 25IVVEYCEPC 1.500 15 88 DLIEAIRRA 1.500 16 37 ATYLELASA 1.000 17 45AVKEQYPGI 0.750 18 97 SNGETLEKI 0.750 19 106 TNSRPPCVI 0.750 20 29YCEPCGFEA 0.500 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected Cw_0301 length selected for subsequencesto be scored 10 echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 44 SAVKeQYPGI50.000 2 33 CGFEaTYLEL 45.000 3 69 INGQlVFSKL 12.000 4 81 GGFPyEKDLI3.750 5 106 TNSRpPCVIL 3.000 6 29 YCEPcGFEAT 2.500 7 16 EVEPgSGVRI 2.5008 65 FEIEiNGQLV 2.160 9 31 EPCGfEATYL 2.000 10 64 AFEIeINGQL 2.000 11 53IEIEsRLGGT 1.500 12 83 FPYEkDLIEA 1.500 13 76 SKLEnGGFPY 1.500 14 21SGVRiVVEYC 1.500 15 37 ATYLeLASAV 1.200 16 80 NGGFpYEKDL 1.200 17 50YPGIeIESRL 1.200 18 93 IRRAsNGETL 1.152 19 23 VRIVvEYCEP 1.000 20 8TSVApPPEEV 1.000 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected Cw_0401 length selected for subsequencesto be scored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 34 GFEATYLEL240.000 2 38 TYLELASAV 30.000 3 82 GFPYEKDLI 25.000 4 18 EPGSGVRIV20.000 5 31 EPCGFEATY 12.000 6 81 GGFPYEKDL 4.800 7 107 NSRPPCVIL 4.8008 70 NGQLVFSKL 4.400 9 75 FSKLENGGF 2.000 10 97 SNGETLEKI 1.584 11 64AFEIEINGQ 1.000 12 84 PYEKDLIEA 1.000 13 49 QYPGIEIES 1.000 14 21SGVRIVVEY 1.000 15 2 SGEPGQTSV 0.660 16 28 EYCEPCGFE 0.600 17 45AVKEQYPGI 0.600 18 9 SVAPPPEEV 0.600 19 105 ITNSRPPCV 0.550 20 77KLENGGFPY 0.500 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected Cw_0401 length selected for subsequencesto be scored 10 echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 64 AFEIEeINGQL200.000 2 74 VFSKlENGGF 100.000 3 50 YPGIeIESRL 80.000 4 31 EPCGfEATYL80.000 5 18 EPGSgVRIVV 10.000 6 34 GFEAtYLELA 10.000 7 28 EYCEpCGFEA6.000 8 33 CGFEaTYLEL 5.760 9 84 PYEKdLIEAI 5.000 10 83 FPYEkDLIEA 4.80011 69 INGQlVFSKL 4.400 12 80 NGGFpYEKDL 4.000 13 106 TNSRpPCVIL 4.000 1456 ESRLgGTGAF 2.000 15 66 EIEInGQLVF 2.000 16 26 VVEYcEPCGF 2.000 17 96ASNGeTLEKI 1.320 18 49 QYPGiEIESR 1.100 19 20 GSGVrIVVEY 1.000 20 38TYLElASAVK 0.792 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected Cw_0602 length selected for subsequencesto be scored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 85 YEKDLIEAI6.600 2 65 FEIEINGQL 6.600 3 21 SGVRIVVEY 6.000 4 31 EPCGFEATY 3.300 561 GTGAFEIEI 3.000 6 38 TYLELASAV 3.000 7 18 EPGSGVRIV 2.420 8 81GGFPYEKDL 2.200 9 94 RRASNGETL 2.200 10 97 SNGETLEKI 2.000 11 70NGQLVFSKL 2.000 12 34 GFEATYLEL 2.000 13 107 NSRPPCVIL 2.000 14 105ITNSRPPCV 1.100 15 47 KEQYPGIEI 1.100 16 66 EIEINGQLV 1.100 17 42LASAVKEQY 1.100 18 77 KLENGGFPY 1.100 19 15 EEVEPGSGV 1.000 20 45AVKEQYPGI 1.000 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected Cw_0702 length selected for subsequencesto be scored 9 echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 31 EPCGFEATY24.000 2 21 SGVRIVVEY 19.200 3 42 LASAVKEQY 8.800 4 77 KLENGGFPY 4.000 549 QYPGIEIES 2.880 6 57 SRLGGTGAF 2.400 7 18 EPGSGVRIV 2.400 8 94RRASNGETL 2.400 9 85 YEKDLIEAI 1.478 10 34 GFEATYLEL 1.440 11 38TYLELASAV 1.440 12 70 NGQLVFSKL 1.440 13 65 FEIEINGQL 1.200 14 81GGFPYEKDL 1.008 15 67 IEINGQLVF 1.000 16 97 SNGETLEKI 0.960 17 61GTGAFEIEI 0.960 18 107 NSRPPCVIL 0.840 19 22 GVRIVVEYC 0.800 20 35FEATYLELA 0.800 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested20 HLA molecule type selected Cw_0702 length selected for subsequencesto be scored 10 echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 106 number of top-scoring subsequencesreported 20 back in scoring output table Scoring Results Score (Estimateof Half Time of Disassociation of a Molecule Subsequence ContainingStart Residue This Rank Position Listing Subsequence 1 20 GSGVrIVVEY38.400 2 30 CEPCgFEATY 16.000 3 41 ELASaVKEQY 16.000 4 50 YPGIeIESRL7.920 5 76 SKLEnGGFPY 4.000 6 69 INGQlVFSKL 2.880 7 18 EPGSgVRIVV 2.4008 33 CGFEaTYLEL 1.440 9 80 NGGFpYEKDL 1.440 10 56 ESRLgGTGAF 1.200 11 93IRRAsNGETL 1.200 12 64 AFEIeINGQL 1.200 13 66 EIEInGQLVF 1.000 14 35FEATyLELAS 0.960 15 87 KDLIeAIRRA 0.800 16 97 SNGEtLEKIT 0.800 17 17VEPGsGVRIV 0.800 18 21 SGVRiVVEYC 0.800 19 28 EYCEpCGFEA 0.720 20 48EQYPgIEIES 0.672 Echoed User Peptide Sequence (length = 115 residues)

TABLE 3 File Name: C35 Prediction Parameters:Quantitative Threshold [%]: 3Inhibitor Threshold [log of fold change]: −1Inhibitor Residues [number]: 1 0--------30---- DRB1*0101:SGVRIVVEYCEPCGF (amino acids 21-35 of SEQ ID NO: 2) DRB1*0301:SGVRIVVEYCEPCGF DRB1*0401: SGVRIVVEYCEPCGF DRB1*0701: SGVRIVVEYCEPCGFDRB1*0801: SGVRIVVEYCEPCGF DRB1*1101: SGVRIVVEYCEPCGF DRB1*1501:SGVRIVVEYCEPCGF DRB5*0101: S

PCGF (binding frame for B5*0101 contains 1 inhibitory residue −100 fold)Quantitative Analysis of ‘SGVRIVVEYCEPCGF’(amino acids 21-35 of SEQ ID NO: 2) Threshold (%):10  09  08  07  06  05  04  03  02  01 DRB1*0101XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0102XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0301XXXXXXXXXXXXXXXXXX.................... DRB1*0401XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX DRB1*0402XX.................................... DRB1*0404XXXXXXXXXXXXXXXXXX.................... DRB1*0405XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0410XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*0421XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0701XXXXXXXXXX............................ DRB1*0801XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0802XXXXXX................................ DRB1*0804XXXXXXXXXXXXXX........................ DRB1*0806XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*1101XXXXXXXXXX............................ DRB1*1104XXXXXXXXXX............................ DRB1*1106XXXXXXXXXX............................ DRB1*1107XXXXXXXXXXXXXXXXXX.................... DRB1*1305XXXXXXXXXXXXXXXXXX.................... DRB1*1307XXXXXXXXXX............................ DRB1*1311XXXXXXXXXX............................ DRB1*1321XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*1501XXXXXXXXXX............................ DRB1*1502XXXXXXXXXX............................ DRB5*0101XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ File Name: C35Prediction Parameters: Quantitative Threshold [%]: 3Inhibitor Threshold [log of fold change]: −1Inhibitor Residues [number]: 1 ---60--------70---- DRB1*0101:SRLGGTGAFEIEINGQLVF (amino acids 57-75 of SEQ ID NO: 2) DRB1*0301:SRLGGTGAFEIEINGQLVF DRB1*0401: SRLGGTGA

VF DRB1*0701: SRLGGTGAFEIEINGQLVF DRB1*0801: SRLGGTGAFEIEINGQLVFDRB1*1101: SRLGGTGAFEIEINGQLVF DRB1*1501: SRLGGTGAFEIEINGQLVF DRB5*0101:SRLGGTGAFEIEINGQLVF(binding frame for *0401 contains 2 inhibitory residues −10 fold each)Quantitative Analysis of ‘SRLGGTGAFEIEINGQLVF’(amino acids 57-75 of SEQ ID NO: 2) Threshold (%):10  09  08  07  06  05  04  03  02  01 DRB1*0101XXXXXXXXXXXXXXXXXXXXXX................ DRB1*0102XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0301XXXXXXXXXXXXXXXXXXXXXX................ DRB1*0401XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0402XXXXXXXXXX............................ DRB1*0404...................................... DRB1*0405XXXXXXXXXX............................ DRB1*0410XX.................................... DRB1*0421XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0701XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX DRB1*0801...................................... DRB1*0802...................................... DRB1*0804XXXXXX................................ DRB1*0806XXXXXX................................ DRB1*1101XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1104XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1106XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1107XX.................................... DRB1*1305XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*1307XX.................................... DRB1*1311XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1321XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1501XXXXXXXXXXXXXXXXXX.................... DRB1*1502XXXXXXXXXXXXXXXXXX.................... DRB5*0101XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ File Name: C35Prediction Parameters: Quantitative Threshold [%]: 3Inhibitor Threshold [log of fold change]: −1Inhibitor Residues [number]: 1 -------70--------80-- DRB1*0101:GAFEIEINGQLVFSKLENGGF (amino acids 63-83 of SEQ ID NO: 2) DRB1*0301:GAFEIEINGQLVFSKLENGGF DRB1*0401: GA

FSKLENGGF DRB1*0701: GAFEIEINGQLVFSKLENGGF DRB1*0801:GAFEIEINGQLVFSKLENGGF DRB1*1101: GAFEIEINGQLVFSKLENGGF DRB1*1501:GAFEIEINGQLVFSKLENGGF DRB5*0101: GAFEIEINGQLVFSKLENGGF(binding frame for *0401 contains 2 inhibitory residues −10 fold each)Quantitative Analysis of ‘GAFEIEINGQLVFSKLENGGF’(amino acids 63-83 of SEQ ID NO: 2) Threshold (%):10  09  08  07  06  05  04  03  02  01 DRB1*0101XXXXXXXXXXXXXXXXXX.................... DRB1*0102XXXXXXXXXXXXXX........................ DRB1*0301XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0401XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0402XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0404XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0405XXXXXXXXXX............................ DRB1*0410XXXXXX................................ DRB1*0421XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0701XXXXXXXXXXXXXXXXXX.................... DRB1*0801...................................... DRB1*0802...................................... DRB1*0804XXXXXX................................ DRB1*0806XXXXXX................................ DRB1*1101XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1104XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1106XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1107XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*1305XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*1307XXXXXXXXXXXXXXXXXX.................... DRB1*1311XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1321XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1501XXXXXXXXXXXXXX........................ DRB1*1502XXXXXXXXXXXXXX........................ DRB5*0101XXXXXXXXXXXXXXXXXXXXXXXXXX............ File Name: C35Prediction Parameters: Quantitative Threshold [%]: 5Inhibitor Threshold [log of fold change]: −1Inhibitor Residues [number]: 1 -------90--------100- DRB1*0101:FPYEKDLIEAIRRASNGETLE (amino acids 83-103 of SEQ ID NO: 2) DRB1*0301:FPYEKDLIEAIRRASNGETLE DRB1*0401: FPYEKDLIEA

LE DRB1*0701: FPYEKDLIEAIRRASNGETLE DRB1*0801: FPYEKDLIEAIRRASNGETLEDRB1*1101: FPYEKDLIEAIRRASNGETLE DRB1*1501: FPYEKDLIEAIRRASNGETLEDRB5*0101: FPYEKDLIEAIRRASNGETLEQuantitative Analysis of ‘FPYEKDLIEAIRRASNGETLE’(amino acids 83-103 of SEQ ID NO: 2) Threshold (%):10  09  08  07  06  05  04  03  02  01 DRB1*0101XXXXXXXXXXXXXXXXXX.................... DRB1*0102XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0301XXXXXXXXXXXXXXXXXXXXXX................ DRB1*0401XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0402XXXXXXXXXXXXXXXXXX.................... DRB1*0404XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0405XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0410XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*0421XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0701XXXXXXXXXX............................ DRB1*0801XXXXXXXXXXXXXXXXXX.................... DRB1*0802XXXXXXXXXXXXXXXXXX.................... DRB1*0804XXXXXXXXXXXXXXXXXXXXXX................ DRB1*0806XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1101XXXXXXXXXX............................ DRB1*1104XXXXXXXXXXXXXXXXXX.................... DRB1*1106XXXXXXXXXXXXXXXXXX.................... DRB1*1107XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1305XXXXXX................................ DRB1*1307XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1311XXXXXXXXXXXXXXXXXX.................... DRB1*1321XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1501XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*1502XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB5*0101XXXXXXXXXXXXXX........................ IMPORTANT NOTE: Tepitope wasprogrammed to evaluate Cys residues as Ala, sence for synthesis andassay limitations it was not possible to systematically test peptidescontaining Cys. So, whenever the predicted sequences contain Cysresidues, we suggest you should have them synthesized REPLACING Cys WITHAla RESIDUES.Altered Peptide Ligands

Identification of immunodominant epitopes of C35 for MHC class Iantigens using specific human T cell lines is a key step toward theirsuccessful use in cancer vaccines. Modified C35 peptide epitopescontaining amino acid substitutions at MHC binding residues have thepotential to be used for enhancement of immune function. Such alteredpeptide ligand, or heteroclitic peptides, can become strong T cellagonists even at 100-fold lower concentrations that the original peptide(Dressel, A. et al., “Autoantigen recognition by human CD8 T Cellclones: enhanced agonist response induced by altered peptide ligand,” J.Immunol. 159:4943-51 (1997). These altered peptide ligand can be of twoforms: those modifications that enhance T cell receptor contact with thepeptide (must be determined experimentally) and those that enhance HLAbinding of the peptide by improving the anchor residues. Table 4specifies modifications that enhance HLA Class I binding by introducingfavorable anchor residues or replacing deleterious residues.

TABLE 4 Modifications that Enhance HLA Class I Binding (Unless otherwiseindicated, examples apply to peptides of 9 amino acids; for 10-mers theamino acid at position 5 is disregarded and the resultant 9-mer isevaluated (http://bimas.dcrt.nih.gov/cgi-bin/molbio/hla_coefficientviewing_page. The modifications listed below are provided by way ofexample based on current data in existing databases and are not intendedin any way to be an inclusive list of all potential alterations ofpeptides binding all potential HLA molecules, both known and unknown todate.) HLA A*0101 Any altered peptide that has S or T at position 2 Anyaltered peptide that has D or E at position 3 Any altered peptide thathas P at position 4 Any altered peptide that has A, F, I, L, M, P, V, orY at position 7 Any altered peptide that has F, K, R, or Y at anchorposition 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: D, E, F, G, H, K, M, N, P,Q, R, W, Y P3: E, K, R, W P4: K, R P7: D, E, G, R P9: D, E, P HLA A*0201Any altered peptide that has F, I, K, L, M, V, W, or Y at position 1 Anyaltered peptide that has I, L, M, Q, or V at anchor position 2 Anyaltered peptide that has F, L, M, W, or Y at position 3 Any alteredpeptide that has D or E at position 4 Any altered peptide that has F atposition 5 Any altered peptide that has F, I, L, M, V, W or Y atauxiliary anchor position 6 Any altered peptide that has F, or W atposition 7 Any altered peptide that has F, W, or Y at position 8 Anyaltered peptide that has I, L, T or V at anchor position 9 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: D, E, H, P P2: C, F, H, K, N, P, R, S, W, Y P3: D, E, K, RP7: D, E, G, R P8: I, V P9: D, E, F, G, H, K, N, P, Q, R, S, W, YHLA-A*0205 Any altered peptide that has F, I, K, L, M, V, W, or Y atposition 1 Any altered peptide that has E, I, L, M, Q, or V at anchorposition 2 Any altered peptide that has F, L, M, W, or Y at position 3Any altered peptide that has D or E at position 4 Any altered peptidethat has F, Y at position 5 Any altered peptide that has F, I, L, M, V,W or Y at auxiliary anchor position 6 Any altered peptide that has F, orW at position 7 Any altered peptide that has F, W, or Y at position 8Any altered peptide that has I, L, T or V at anchor position 9 Anyaltered peptide where deleterious residues at the following positionsare replaced: P1: D, E, P P2: C, D, F, G, H, K, N, P, R, S, W, Y P3: D,E, K, R P7: D, E, R P9: D, E, F, G, H, K, N, P, Q, R, S, W, Y HLA-A*03Any altered peptide that has G or K at position 1 Any altered peptidethat has I, L, M, Q, T or V at anchor position 2 Any altered peptidethat has F, I, L, M, V, W, or Y at position 3 Any altered peptide thathas E, G or P at position 4 Any altered peptide that has F, I, P, V, W,Y at position 5 Any altered peptide that has F, I, L, M, or V atposition 6 Any altered peptide that has F, I, L, M, W, or Y at position7 Any altered peptide that has F, I, K, L, Q or Y at anchor position 9Any altered peptide where deleterious residues at the followingpositions are replaced: P1: D, E, P P2: D, E, F, G, H, K, N, R, S, W, YP7: G, K, R P9: D, E, G, H, N, P, Q, S, T HLA-A*1101 Any altered peptidethat has G, K or R at position 1 Any altered peptide that has I, L, M,Q, T, V, Y at anchor position 2 Any altered peptide that has F, I, L, M,V, W, Y at position 3 Any altered peptide that has F, I, L, M, W or Y atposition 7 Any altered peptide that has K or R at anchor position 9 Anyaltered peptide where deleterious residues at the following positionsare replaced: P1: D, E, P P2: D, E, G, H, K, N, R, S, W P7: K, R P9: C,D, E, G, N, P, Q, S, T HLA-A24 Any altered peptide that has K or R atposition 1 Any altered peptide that has F or Y at anchor position 2 Anyaltered peptide that has E, I, L, M, N, P, Q, or Vat position 3 Anyaltered peptide that has D, E, or P at position 4 Any altered peptidethat has I, L, or V at position 5 Any altered peptide that has F atposition 6 Any altered peptide that has N or Q at position 7 Any alteredpeptide that has E or K at position 8 Any altered peptide that has F, I,L, or M at anchor position 9 Any altered peptide where deleteriousresidues at the following positions are replaced: P1: P P2: D, E, H, K,R P9: D, E, G, H, K, P, Q, R HLA-A*3101 Any altered peptide that has Kor R at position 1 Any altered peptide that has F, I, L, M, Q, T, V, orY at anchor position 2 Any altered peptide that has F, I, L, M, V W, orY at position 3 Any altered peptide that has F, I, L, M, or V atposition 6 Any altered peptide that has F, I, L, M, W, or Y at position7 Any altered peptide that has K or R at anchor position 9 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: D, E, P P2: D, E, G, H, K, N, R, S P7: K, R P9: C, G, N,P, Q, S, T HLA-A*3302 Any altered peptide that has D or E at position 1Any altered peptide that has I, L, M, S, V or Y at anchor position 2 Anyaltered peptide that has R at anchor position 9 Any altered peptidewhere deleterious residues at the following positions are replaced: P1:K, P, R P2: D, E, K, R P9: D, E, F, G, N, P, W, Y HLA-B7 Any alteredpeptide that has A at position 1 Any altered peptide that has A, P or Vat anchor position 2 Any altered peptide that has M or R at position 3Any altered peptide that has P at position 5 Any altered peptide thathas R at position 6 Any altered peptide that has I, L, M or V at anchorposition 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: D, E, F, H, K, R, W, Y P3:D, E P9: D, E, F, G, H, K, N, P, Q, R, S, W, Y HLA-B8 Any alteredpeptide that has D or E at position 1 Any altered peptide that has A, C,L, or P at anchor position 2 Any altered peptide that has K or R atposition 3 Any altered peptide that has D or E at position 4 Any alteredpeptide that has K or R at position 5 Any altered peptide that has I, L,M, or V at anchor position 9 Any altered peptide where deleteriousresidues at the following positions are replaced: P1: K, P, R P2: D, E,F, G, H, K, Q, R, W, or Y P3: D, E P5: D, E P9: D, E, F, G, H, K, N, P,Q, R, S, W, Y HLA-B8 (8-mer peptides) Any altered peptide that has D orE at position 1 Any altered peptide that has A, C, L, or P at anchorposition 2 Any altered peptide that has K or R at position 3 Any alteredpeptide that has D or E at position 4 Any altered peptide that has K orR at position 5 Any altered peptide that has I, L, M, or V at anchorposition 8 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: K, P, R P2: D, E, F, G, H, K, Q,R, W, or Y P3: D, E P5: D, E P8: D, E, F, G, H, K, N, P, Q, R, S, W, YHLA-B14 Any altered peptide that has D or E at position 1 Any alteredpeptide that has K or R at anchor position 2 Any altered peptide thathas F, I, L, M, P, V, W, Y at position 3 Any altered peptide that has Hor R at position 5 Any altered peptide that has I, L, M, R, or V atposition 6 Any altered peptide that has T at position 7 Any alteredpeptide that has I, L, M, or V at anchor position 9 Any altered peptidewhere deleterious residues at the following positions are replaced: P1:P P2: D, E, F, W, or Y P3: E, R P5: E, W, Y P9: D, E, G, H, K, N, P, Q,R HLA-B*2702 Any altered peptide that has K or R at position 1 Anyaltered peptide that has E, L, M, N, Q or R at anchor position 2 Anyaltered peptide that has F, W, or Y at position 3 Any altered peptidethat has F, I, L, W or Y at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: D, E,P P2: D, F, G, H, K, W, or Y P7: K P9: D, E, G, K, N, P, Q, R, SHLA-B27*05 (8-mer peptides) Any altered peptide that has K or R atposition 1 Any altered peptide that has E, L, M, N, Q or R at anchorposition 2 Any altered peptide that has F, W, or Y at position 3 Anyaltered peptide that has F, I, K, L, M, R, V or Y at anchor position 8Any altered peptide where deleterious residues at the followingpositions are replaced: P1: D, E, P P2: D, F, G, H, K, W, or Y P7: K P9:D, E, G, K, N, P, Q, R, S HLA-B*3501 (8-mer peptides) Any alteredpeptide that has K or R at position 1 Any altered peptide that has A, P,or S at anchor position 2 Any altered peptide that has K or R atposition 3 Any altered peptide that has D or E at position 4 Any alteredpeptide that has D or E at position 5 Any altered peptide that has F, I,L, M, V, W or Y at anchor position 8 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P2:D, E, F, H, K, R, W, Y P3: D, E P8: D, E, F, G, H, K, P, Q, R HLA-B*3701Any altered peptide that has D or E at anchor position 2 Any alteredpeptide that has I or V at position 5 Any altered peptide that has F, L,or M at position 8 Any altered peptide that has F, I, L, M, V or Y atanchor position 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P9: D, E, G, H, K, P, Q, RHLA-B*3801 Any altered peptide that has F, H, P, W or Y at anchorposition 2 Any altered peptide that has D or E at position 3 Any alteredpeptide that has D, E, or G at position 4 Any altered peptide that hasA, I, L, M, or V at position 5 Any altered peptide that has K or Y atposition 8 Any altered peptide that has F, I, L, M, or V at anchorposition 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: D, E, K, R P3: K, R P9: D,E, G, H, K, P, Q, R HLA-B*3901 (8-mer peptides) Any altered peptide thathas H or R at anchor position 2 Any altered peptide that has D, E, F, I,L, M, V, W, or W at position 3 Any altered peptide that has D or E atposition 4 Any altered peptide that has I, L, M, or V at position 6 Anyaltered peptide that has I, L, M or V at anchor position 8 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: P P2: D, E P3: K, R P6: D, E, K, R P8: D, E, G, H, K, P,Q, R HLA-B*3902 Any altered peptide that has K or Q at anchor position 2Any altered peptide that has F, I, L, M, V, W, or Y at position 5 Anyaltered peptide that has F, L, or M at anchor position 9 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: P P2: D, E P3: K, R P9: D, E, G, H, K, P, Q, R HLA-B40 Anyaltered peptide that has A or G at position 1 Any altered peptide thathas D or E at anchor position 2 Any altered peptide that has A, F, I, L,M, V, W, or Y at position 3 Any altered peptide that has P at position 4Any altered peptide that has P at position 5 Any altered peptide thathas A, L, M, or W at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P2:F, H, I, K, L, M, Q, R, V, W, or Y P3: D, E, K, R P9: D, E, G, H, K, N,P, Q, R HLA-B44*03 Any altered peptide that has A, D, or S at position 1Any altered peptide that has D or E at anchor position 2 Any alteredpeptide that has A, I, L, M, or V at position 3 Any altered peptide thathas F, I, or P at position 4 Any altered peptide that has A, K, or V atposition 5 Any altered peptide that has A, L, T, or V at position 6 Anyaltered peptide that has F, K, or T at position 7 Any altered peptidethat has K at position 8 Any altered peptide that has F, W or Y atanchor position 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: F, H, I, K, L, M, Q, R, V,W, Y P9: D, E, G, H, K, N, P, Q, R HLA-B*5101 (8-mer peptides) Anyaltered peptide that has D, E, F, I, L, M, V, or Y at position 1 Anyaltered peptide that has A, G or P at anchor position 2 Any alteredpeptide that has F, W or Y at position 3 Any altered peptide that has D,E, G, I, K, or V at position 4 Any altered peptide that has A, G, I, S,T, or V at position 5 Any altered peptide that has I, K, L, N, or Q atposition 6 Any altered peptide that has D, K, Q, or R at position 7 Anyaltered peptide that has I, L, M, or V at anchor position 8 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: K, P, R P2: D, E, H, K P8: D, E, F, G, H, K, N, P, Q, R,S, W, Y HLA-B*5102 Any altered peptide that has F or Y at position 1 Anyaltered peptide that has A, G, or P at anchor position 2 Any alteredpeptide that has F, I, L, V, W, or Y at position 3 Any altered peptidethat has E, G, H, K, L, N, Q, R, or T at position 4 Any altered peptidethat has G, N, Q, T, or V at position 5 Any altered peptide that has I,N, Q, or T at position 6 Any altered peptide that has E, K, Q, or R atposition 7 Any altered peptide that has K, R, T, or Y at position 8 Anyaltered peptide that has I, L, M, or V at anchor position 9 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: P P2: D, E, H, K, R P3: D, E, K, R P9: D, E, F, G, H, K,N, P, Q, R, S, W, Y HLA-B*5102 (8-mer peptides) Any altered peptide thathas F or Y at position 1 Any altered peptide that has A, G, or P atanchor position 2 Any altered peptide that has F, I, L, V, W, or Y atposition 3 Any altered peptide that has E, G, H, K, L, V, W, or Y atposition 4 Any altered peptide that has G, N, Q, T, V at position 5 Anyaltered peptide that has I, N, or Q at position 6 Any altered peptidethat has Q, or R at position 7 Any altered peptide that has I, L, M, orV at position 8 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: D, E, H, K, R P3: D, E, K, RP8: D, E, F, G, H, K, N, P, Q, R, S, W, Y HLA-B*5103 Any altered peptidethat has D, T, or V at position 1 Any altered peptide that has A, G, orP at anchor position 2 Any altered peptide that has D, F, L, or Y atposition 3 Any altered peptide that has E, G, L, N, Q, R, T, or V atposition 4 Any altered peptide that has A, G, M, N, Q, R, K or V atposition 5 Any altered peptide that has I, K, or T at position 6 Anyaltered peptide that has M or V at position 7 Any altered peptide thathas I, L, M, or V at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P2:D, E, H, K, R P9: D, E, F, G, H, K, N, P, Q, R, S, W, Y HLA-B*5201(8-mer peptides) Any altered peptide that has I, L, M, or V at position1 Any altered peptide that has G, P, or Q at anchor position 2 Anyaltered peptide that has D, F, I, L, P, W, or Y at position 3 Anyaltered peptide that has A, E, I, K, L, P, or V at position 4 Anyaltered peptide that has A, F, G, I, L, M, T or V at position 5 Anyaltered peptide that has K, L, N, S or T at position 6 Any alteredpeptide that has E, K, Q, or Y at position 7 Any altered peptide thathas F, I, L, M, or V at anchor position 8 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P2:H, K, R P3: R P8: D, E, G, H, K, N, P, Q, R, S HLA-B*5801 Any alteredpeptide that has I, K, or R at position 1 Any altered peptide that hasA, S, or T at anchor position 2 Any altered peptide that has D atposition 3 Any altered peptide that has E, K, or P at position 4 Anyaltered peptide that has F, I, L, M, or V at position 5 Any alteredpeptide that has F, I, L, or V at position 6 Any altered peptide thathas L, M, N, or Y at position 7 Any altered peptide that has K, N, R, orT at position 8 Any altered peptide that has F, W, or Y at anchorposition 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: D, E, P P2: D, E, F, H, I, K, L,M, N, Q, R, V, W, Y P9: D, E, G, H, K, N, P, Q, R, S HLA-B*60 Anyaltered peptide that has D or E at anchor position 2 Any altered peptidethat has A, I, L, M, S, or V at position 3 Any altered peptide that hasL, I, or V at position 5 Any altered peptide that has I, L, M, V, or Yat position 7 Any altered peptide that has K, Q, or R at position 8 Anyaltered peptide that has I, L, M, or V at anchor position 9 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: P P2: F, H, I, K, L, M, Q, R, V, W, Y P9: D, E, F, G, H,K, N, P, Q, R, S, W, Y HLA-B*61 Any altered peptide that has G or R atposition 1 Any altered peptide that has D or E at anchor position 2 Anyaltered peptide that has A, F, I, L, M, T, V, W, or Y at position 3 Anyaltered peptide that has I at position 6 Any altered peptide that has Yat position 7 Any altered peptide that has A, I, L, M, or V at anchorposition 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: F, H, I, K, L, M, Q, R, V,W, Y P9: D, E, F, G, H, K, N, P, Q, R, S, W, Y HLA-B*61 (8-mer peptides)Any altered peptide that has G or R at position 1 Any altered peptidethat has D or E at anchor position 2 Any altered peptide that has A, F,I, L, M, T, V, W, or Y at position 3 Any altered peptide that has I atposition 6 Any altered peptide that has Y at position 7 Any alteredpeptide that has A, I, L, M, or V at anchor position 8 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: P P2: F, H, I, K, L, M, Q, R, V, W, Y P8: D, E, F, G, H,K, N, P, Q, R, S, W, Y HLA-B*62 Any altered peptide that has I atposition 1 Any altered peptide that has I, L, Q at anchor position 2 Anyaltered peptide that has G, K, R at position 3 Any altered peptide thathas D, E, G, or P at position 4 Any altered peptide that has F, G, I, L,or V at position 5 Any altered peptide that has I, L, T, V at position 6Any altered peptide that has T, V, or Y at position 7 Any alteredpeptide that has F, W, Y at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P2:D, E, F, H, K, N, R, S, W, Y P3: D, E P6: D, E, K, R P9: D, E, G, H, K,N, P, Q, R, S HLA-Cw0301 Any altered peptide that has A or R at anchorposition 2 Any altered peptide that has F, I, L, M, V, or Y at position3 Any altered peptide that has E, P, or R at position 4 Any alteredpeptide that has N at position 5 Any altered peptide that has F, M, or Yat position 6 Any altered peptide that has K, M, R, or S at position 7Any altered peptide that has T at position 8 Any altered peptide thathas F, I, L, M at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P3:D, K, R P6: D, E, K, R P9: D, E, G, H, K, N, P, Q, R, S, HLA-Cw0401 Anyaltered peptide that has F, P, W, or Y at anchor position 2 Any alteredpeptide that has D, or H at position 3 Any altered peptide that has D orE at position 4 Any altered peptide that has A, H, M, R, or T atposition 5 Any altered peptide that has I, L, M, or V at position 6 Anyaltered peptide that has A at position 7 Any altered peptide that has H,K, or S at position 8 Any altered peptide that has F, I, L, M, V or Y atanchor position 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: D, E, H, K, R P9: D, E, G,H, K, N, P, Q, R, S HLA-Cw0602 Any altered peptide that has F, I, K, orY at position 1 Any altered peptide that has A, P, Q, or R at anchorposition 2 Any altered peptide that has F, I, K, L, or M at position 5Any altered peptide that has I, L, or V at position 6 Any alteredpeptide that has K, N, Q, or R at position 7 Any altered peptide thathas I, L, M, V, or Y at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P9:D, E, G, H, K, N, P, Q, R, S

Examples of predicted human Class I MHC binding peptides from the C35 aasequence and how they might be changed to improve binding (SEQ ID NO: isshown in parentheses unless peptide is a subsequence of SEQ ID NO:2):

SEQ ID NO: 2 Rank Start position SubsequenceScore (estimated half time of dissociation) HLA-A*0101 1 77 KLENGGRPY225.000 2 16 EVEPGSGVR 90.000 3 29 YCEPCGFEA 45.000 4 39 YLELASAVK36.000 5 2 SGEPGQTSV 2.250 G is deleterious at P2 example ofSTEPGQTSV (2059) 22.50 improved peptide G replaced with T @ P2example of STEPGQISY (2060) 5625.00 improved peptideV at P9 replaced with Y, P7 enhanced HLA-A*0101 (10-mer peptides) 1 66EIEINGQLVF 45.000 2 16 EVEPGSGVRI 18.000 3 29 YCEPCGFEAT 9.000 4 26VVEYCEPCGF 9.000 5 52 GIEIESRLGG 2.250 example of GTEPSRLGY (206 1)1125.000 improved peptide replace I with T @P2 replace G with Y @P9P5 enhanced with P HLA-A*0201 (9-mer peptides) 1 9 SVAPPPEEV 2.982 2 104KITNSRPPC 2.391 3 105 ITNSRPPCV 1.642 4 25 IVVEYCEPC 1.485 5 65FEIEINGQL 1.018 example of FLIEINWYL (2062) 16619.000 improved peptideHLA-A*0201 (10-mer peptides) 1 58 RLGGTGAFEI 60.510 2 104 KITNSRPPCV33.472 3 65 FEIEINGQLV 25.506 4 83 FPYEKDLIEA 4.502P is deleterious at P2 example of FLYEKDLIEA (2063) 689.606improved peptide replace P with L @ P2 example of FLYEKDLIEV (2064)9654.485 improved peptide replace A with V @ P9 5 33 CGFEATYLEL 3.173HLA-A*0205 1 65 FEIEINGQL 8.820 2 25 IVVEYCEPC 3.060 3 9 SVAPPPEEV 2.0004 104 KITNSRPPC 1.500 5 81 GGFPYEKDL 1.260 G is deleterious at P2example of GVFPYEKDL (2065) 50.400 improved peptide replace G with V @P2 HLA-A*0205 (10-mer peptides) 1 33 CGEFATYLEL 6.300G is deleterious at P2 example of CVEFATYLEL (2066) 11.200improved peptide replace G with V @ P2 2 104 KITNSRPPCV 6.000 3 65FEIEINGQLV 2.520 4 53 IEIESRLGGT 1.428 5 83 FPYEKDLIEA 1.350P is deleterious at P2 example of FVYEKDLIEA (2067) 54.000improved peptide replace P with V @ P2 HLA-A24 1 34 GFEATYLEL 33.000 249 QYPGIEIES 11.550 example of QYPGIEIEL (2068) 462.000 improved peptideenhance P9 3 70 NGQLZFSKL 11.088 4 38 TYLELASAV 10.800 5 82 GFPYEKDLI7.500 HLA-A24 (10-mer peptides) 1 64 AFEIEINGQL 42.000 2 74 VFSKLENGGF10.000 3 84 PYEKDLIEAI 9.000 4 69 INGQLVFSKL 7.392 example ofIYGQLVFSKL (2069) 369.6 improved peptide enhance P2 5 28 EYCEPCGFEA6.600 HLA-A3 1 77 KLENGGFPY 36.000 example of KLENGGFPK (2070) 180.000improved peptide enhance P9 2 39 YLELASAVK 20.000 3 101 TLEKITNSR 6.0004 61 GTGAFEIEI 0.540 5 69 INGQLVFSK 0.360 N is deleterious @ P2example of ILGQLVFSK (2071) 180.000 improved peptide replace N with L @P2 HLA-A3 (10-mer peptides) 1 68 EINGQLVFSK 8.100 2 58 RLGGTGAFEI 2.7003 41 ELASAVKEQY 1.800 4 78 LENGGFPYEK 0.810 E is deleterious @ P2example of LLNGGFPYEK (2072) 270.000 improved peptide replace E with L @P2 5 95 RASNGETLEK 0.400 HLA-A*1101 1 39 YLELASAVK 0.400 2 69 INGQLVFSK0.120 N is deleterious @ P2 example of IVGQLVFSK (2073) 6.000improved peptide replace N with V @ P2 3 16 EVEPGSGVR 0.120 4 101TLEKITNSR 0.080 5 61 GTGAFEIEI 0.060 HLA-A*1101 (10-mer peptides) 1 95RASNGETLEK 1.200 2 38 TYLELASAVK 0.600 3 68 EINGGLVFSK 0.360 4 78LENGGFPYEK 0.120 E is deleterious @ P2 example of LVNGGFPYEK (2074)4.000 improved peptide replace E with V @ P2 5 100 ETLEKITNSR 0.090HLA-A*3101 1 101 TLEKITNSR 2.000 2 16 EVEPGSGVR 0.600 3 50 YPGIEIESR0.400 4 87 KDLIEAIRR 0.240 D is deleterious @ P2 example ofKILIEAIRR (2075) 12.000 improved peptide replace D with I @ P2 5 39YLELASAVK 0.200 HLA-A*3302 1 16 EVEPGSGVR 45.000 2 101 TLEKITNSR 9.000 350 YPGIEIESR 3.000 4 66 EIEINGQLV 1.500 5 56 ESRLGGTGA 1.500HLA-A*3302 (10-mer peptides) 1 49 QYPGIEIESR 15.000 2 100 ETLEKITNSR9.000 3 16 EVEPGSGVRI 1.500 4 28 EYCEPCGFEA 1.500 5 68 EINGQLVFSK 1.500HLA-A68.1 1 16 EVEPGSGVR 900.000 2 9 SVAPPPEEV 12.000 3 50 YPGIEIESR10.000 example of YVGIEIESR (2076) 400.000 improved peptide enhance P2 496 ASNGETLEK 9.000 5 101 TLEKITNSR 5.000 HLA-A68.1 (10-mer peptides) 1100 ETLEKITNSR 300.000 2 16 EVEPGSGVRI 18.000 3 68 EINGGLVFSK 9.000 4 15EEVEPGSGVR 9.000 E is deleterious @ P2 example of EVVEPGSGR (2077)1200.00 improved peptide replace E with V @ P2 5 95 RASNGETLEK 3.000HLA-B14 1 94 RRASNGETL 20.000 2 57 SRLGGTGAF 5.000 example ofSRLGGTGAL (2078) 100.000 improved peptide enhance P9 3 100 ETLEKITNS3.375 4 105 ITNSRPPCV 2.000 5 88 DLIEAIRRA 1.350HLA-B14 (10-mer peptides) 1 103 EKITNSRPPC 6.750 example ofERITNSRPPL (2079) 900.000 improved peptide enhance P10 2 33 CGFEATYLEL5.000 3 93 IRRASNGETL 4.000 4 18 EPGSGVRIVV 3.000 5 88 DLIEAIRRAS 2.250HLA-B40 1 65 FEIEINGQL 80.000 2 3 GEPGQTSVA 40.000 3 35 FEATYLELA 40.0004 15 EEVEPGSGV 24.000 example of EEVEPGSGL (2080) 120.000improved peptide enhance P9 5 67 IEINGQLVF 16.000HLA-B40 (10-mer peptides) 1 55 IESRLGGTGA 20.000 2 53 IEIESRLGGT 16.000example of IEIESRLGGL (2081) 80.000 improved peptide enhance P10 3 65FEIEINGQLV 16.000 4 67 IEINGQLVFS 16.000 5 99 GETLEKITNS 8.000 HLA-B60 165 FEIEFNGQL 387.200 2 17 VEPGSGVRI 17.600 example of VEPGSGVRL (2082)352.000 improved peptide enhance P9 3 15 EEVEPGSGV 16.000 4 47 KEQYPGIEI16.000 5 85 YEKDLIEAI 8.800 HLA-B60 (10-mer peptides) 1 65 FEIEINGQLV16.000 example of FEIEINGQLL (2083) 320.000 improved peptide enhance P102 106 TNSRPPCVIL 16.000 3 53 IEIESRLGGT 8.000 4 33 CGFEATYLEL 8.000 5 17VEPGSGVRIV 8.000 HLA-B61 1 15 EEVEPGSGV 80.000 2 35 FEATYLELA 40.000example of FEATYLELV (2084) 160.000 improved peptide enhance P9 3 3GEPGQTSVA 22.000 4 65 FEIEINGQL 16.000 5 85 YEKDLIEAI 16.000HLA-B61 (10-mer peptides) 1 65 FEIEINGQLV 80.000 2 17 VEPGSGVRIV 40.0003 55 IESRLGGTGA 20.000 4 87 KDLIEAIRRA 10.000 example ofKELIEAIRRV (2085) 160.000 improved peptide enhance P2, P10 5 53IEIESRLGGT 8.000 HLA-B62 1 77 KLENGGFPY 24.000 2 21 SGVRIVVEY 4.800 3 75FSKLENGGF 3.000 4 31 EPCGFEATY 2.640 P is deleterious @ P2 example ofEQCGFEATY (2086) 105.6 improved peptide replace P with Q @ P2 5 88DLIEAIRRA 2.200 HLA-B62 (10-mer peptides) 1 41 ELASAVKEQY 40.000 2 58RLGGTGAFEI 9.600 3 66 EIEINGQLVF 7.920 4 56 ESRLGGTGAF 6.000S is deleterious @ P2 example of EQRLGGTGAF (2087) 480.000improved peptide replace S with Q @ P2 5 20 GSGVRIVVEY 4.800S is deleterious @ P2 example of GQGVRIVVEY (2088) 384.000improved peptide replace S with Q @P2 HLA-B7 1 107 NSRPPCVIL 60.000example of NPRPPCVIL (2089) 1200.000 improved peptide enhance P2 2 45AVKEQYPGI 6.000 3 22 GVRIVVEYC 5.000 4 70 NGQLVFSKL 4.000 5 81 GGFPYEKDL4.000 HLA-B7 (10-mer peptides) 1 50 YPGIEIESRL 80.000 2 31 EPCGFEATYL80.000 3 18 EPGSGVRIVV 6.000 example of EPGSGVRIVL (2090) 120.000improved peptide enhance P10 4 106 TNSRPPCVIL 6.000 5 80 NGGFPYEKDL4.000 HLA-B8 1 107 NSRPPCVIL 4.000 2 45 AVKEQYPGI 1.500 3 105 ITNSRPPCV0.600 4 56 ESRLGGTGA 0.400 5 100 ETLEKITNS 0.300 S is deleterious @ P9example of ETLEKITNL (2091) 12.000 improved peptide replace S with L @P9 HLA-B8 (8-mer peptides) 1 83 FPYEKDLI 6.000 2 107 NSRPPCVI 1.000 3 91EAIRRASN 0.800 N is deleterious @ P8 example of EAIRRASL (2092) 32.000improved peptide replace N with L @ P9 4 20 GSGVRIVV 0.600 5 18 EPGSGVRI0.400 HLA-B8 (10-mer peptides) 1 50 YPGIEIESRL 0.800 2 93 IRRASNGETL0.400 example of IA RASNGETL (2093) 16.000 improved peptidereplace R with A @ P2 3 31 EPCGFEATYL 0.320 4 104 KITNSRPPCV 0.300 5 18EPGSGVRIVV 0.240 HLA-B*2702 1 57 SRLGGTGAF 200.000 2 94 RRASNGETL180.000 example of RRASNGETF (2094) 600.000 improved peptide enhance P93 93 IRRASNGET 20.000 4 27 VEYCEPCGF 15.000 5 77 KLENGGFPY 9.000HLA-B*2702 (10-mer peptides) 1 93 IRRASNGETL 60.000 2 94 RRASNGETLE6.000 3 30 CEPCGFEATY 3.000 4 58 RLGGTGAFEI 2.700 5 23 VRIVVEYCEP 2.000P is deleterious @ P10 example of VRIVVEYCEY (2095) 200.000improved peptide replace P with Y @ P10 HLA-B*2705 1 94 RRASNGETL6000.000 2 57 SRLGGTGAF 1000.000 3 93 IRRASNGET 200.000 example ofIRRASNGEL (2096) 2000.000 improved peptide enhance P9 4 27 VEYCEPCGF75.000 5 77 KLENGGFPY 45.000 HLA-B*2705 (10-mer peptides) 1 93IRRASNGETL 2000.000 2 94 RRASNGETLE 60.000 E is deleterious @ P2example of RRASNGETLL (2097) 6000.000 improved peptidereplace E with L @ P2 3 78 LENGGFPYEK 30.000 4 95 RASNGETLEK 30.000 5 58RLGGTGAFEI 27.000 HLA-B*3501 1 31 EPCGFEATY 40.000 2 75 FSKLENGGF 22.500example of FPKLENGGM (2098) 120.000 improved peptide enhance P2, P9 3107 NSRPPCVIL 15.000 4 42 LASAVKEQY 6.000 5 18 EPGSGVRIV 4.000HLA-B*3501 (10-mer peptides) 1 31 EPCGFEATYL 30.000 2 50 YPGIEIESRL20.000 3 56 ESRLGGTGAF 15.000 4 20 GSGVRIVVEY 10.000 5 83 FPYEKDLIEA6.000 example of FPYEKDLIEM (2099) 120.000 improved peptide enhance P10HLA-B*3701 1 65 FEIEINGQL 15.000 example of FDIEINGQL (2100) 60.000improved peptide enhance P2 2 47 KEQYPGIEI 10.000 3 85 YEKDLIEAI 10.0004 17 VEPGSGVRI 10.000 5 35 FEATYLELA 5.000 HLA-B*3701 (10-mer peptides)1 65 FEIEINGQLV 10.000 example of FDIEINGQLI (2101) 200.000improved peptide enhance P2, P10 2 67 IEINGQLVFS 5.000 3 81 GGFPYEKDLI5.000 4 87 KDLIEAIRRA 4.000 5 30 CEPCGFEATY 2.000 HLA-B*3801 1 34GFEATYLEL 6.000 example of GHEATYLEL (2102) 90.000 improved peptideenhance P2 2 70 NGQLVFSKL 1.560 3 38 TYLELASAV 1.040 4 81 GGFPYEKDL1.000 5 97 SNGETLEKI 0.720 HLA-B*3801 (10-mer peptides) 1 64 AFEIEINGQL7.800 example of AHEIEINGQL (2103) 117.000 improved peptide enhance P2 231 EPCGFEATYL 4.800 3 66 EIEINGQLVF 3.000 4 26 VVEYCEPCGF 3.000 5 50YPGIEIESRL 2.600 HLA-B*3901 1 94 RRASNGETL 15.000 example ofRHASNGETL (2104) 90.000 improved peptide enhance P2 2 34 GFEATYLEL 9.0003 38 TYLELASAV 4.000 4 66 EIEINGQLV 3.000 5 2 SGEPGQTSV 3.000HLA-B*3901 (10-mer peptides) 1 33 CGFEATYLEL 12.000 example ofCHFEATYLEL (2105) 360.000 improved peptide enhance P2 2 64 AFEIEINGQL9.000 3 93 IRRASNGETL 4.500 4 46 VKEQYPGIEI 3.000 5 16 EVEPGSGVRI 3.000HLA-B*3902 1 70 NGQLVFSKL 2.400 example of PNKQLVFSKL (2106) 24.000improved peptide enhance P2 2 81 GGFPYEKDL 2.400 3 94 RRASNGETL 2.000 434 GFEATYLEL 2.000 5 107 NSRPPCVIL 0.600 HLA-B*3902 (10-mer peptides) 169 INGQLVFSKL 2.400 2 64 AFEIEINGQL 2.400 3 50 YPGIEIESRL 2.400 4 80NGGFPYEKDL 2.400 5 106 TNSRPPCVIL 2.000 HLA-B*4403 1 67 IEINGQLVF200.000 example of IEINGQLVY (2107) 900.000 improved peptide enhance P92 27 VEYCEPCGF 40.000 3 21 SGVRIVVEY 36.000 4 65 FEIEINGQL 20.000 5 35FEATYLELA 12.000 HLA-B*4403 (10-mer peptides) 1 30 CEPCGFEATY 120.000 253 IEIESRLGGT 30.000 example of IEIESRLGGY (2108) 900.000improved peptide enhance P10 3 67 IEINGQLVFS 30.000 4 65 FEIEINGQLV20.000 5 17 VEPGSGVRIV 18.000 HLA-B*5101 1 18 EPGSGVRIV 484.000 2 59LGGTGAFEI 114.400 example of LPGTGAFEI (2109) 572.000 improved peptideenhance P2 3 2 SGEPGQTSV 48.400 4 81 GGFPYEKDL 44.000 5 70 NGQLVFSKL22.000 HLA-B*5101 (10-mer peptides) 1 18 EPGSGVRIVV 440.000 2 44SAVKEQYPGI 220.000 example of SPVKEQYPGI (2110) 440.000 improved peptideenhance P2 3 31 EPCGFEATYL 220.000 4 81 GGFPYEKDLI 176.000 5 50YPGIEIESRL 157.300 HLA-B*5102 1 18 EPGSGVRIV 242.000 2 81 GGFPYEKDL110.000 example of GPFPYEKLDI (2111) 2200.000 improved peptideenhance P2, P9 3 59 LGGTGAFEI 96.800 4 70 NGQLVFSKL 48.400 5 2 SGEPGQTSV24.200 HLA-B*5102 (10-mer peptide) 1 44 SAVKEQYPGI 726.000 example ofSPVKEQYPGI (2112) 1452.000 improved peptide enhance P2 2 50 YPGIEIESRL400.000 3 81 GGFPYEKDLI 400.000 4 18 EPGSGVRIVV 220.000 5 31 EPCGFEATYL121.000 HLA-B*5103 1 59 LGGTGAFEI 48.400 example of LAFTGAFEI (2113)145.200 improved peptide enhance P2 2 2 SGEPGQTSV 44.000 3 18 EPGSGVRIV44.000 4 70 NGQLVFSKL 7.260 5 81 GGFPYEKDL 7.200HLA-B*5103 (10-mer peptide) 1 44 SAVKEQYPGI 110.000 2 81 GGFPYEKDLI52.800 3 18 EPGSGVRIVV 44.000 example of EAGSGVRIVV (2114) 110.000improved peptide enhance P2 4 60 GGTGAFEIEI 44.000 5 33 CGFEATYLEL 7.920HLA-B*5201 1 18 WPGSGVRIV 75.000 2 67 LEINGQLVF 22.500 example ofLQINGQLVI (2115) 450.000 improved peptide enhance P2, P9 3 59 LGGTGAFEI11.250 4 98 NGETLEKIT 11.000 5 19 PGSGVRIVV 10.000HLA-B*5201 (10-mer peptides) 1 18 EPGSGVRIVV 100.000 2 17 VEPGSGVRIV45.000 example of VQPGSGVRIV (2116) 450.000 improved peptide enhance P23 81 GGFPYEKDLI 33.000 4 105 ITNSRPPCVI 15.000 5 37 ATYLELASAV 12.000HLA-B*5801 1 75 FSKLENGGF 40.000 example of FSKLENGGW (2117) 80.000improved peptide enhance P9 2 42 LASAVKEQY 4.500 3 107 NSRPPCVIL 4.000 461 GTGAFEIEI 3.000 5 105 ITNSRPPCV 3.000 HLA-B*5801 (10-mer peptides) 156 ESRLGGTGAF 12.000 2 20 GSGVRIVVEY 10.800 example of GSGVRIVVEW (2118)144.000 improved peptide enhance P10 3 1 MSGEPGQTSV 4.000 4 105ITNSRPPCVI 3.000 5 37 ATYLELASAV 3.000 HLA-Cw*0301 1 65 FEIEINGQL 30.0002 81 GGFPYEKDL 18.000 3 70 NGQLVFSKL 12.000 4 57 SRLGGTGAF 10.000 5 34GFEATYLEL 10.000 HLA-Cw*0301 (10-mer peptides) 1 44 SAVKEQYPGI 50.000example of SAVKEQYPGL (2119) 100.000 improved peptide enhance P10 2 33CGFEATYLEL 45.000 3 69 INGQLVFSKL 12.000 4 81 GGFPYEKDLI 3.750 5 106TNSRPPCVIL 3.000 HLA-Cw*0401 1 34 GFEATYLEL 240.000 2 38 TYLELASAV30.000 3 82 GFPYEKDLI 25.000 4 18 EPGSGVRIV 20.000 5 31 EPCGFEATY 12.000example of EFCGFEATL (2120) 200.000 improved peptide enhance P2, P9HLA-Cw*0401 (10-mer peptides) 1 64 AFEIEINGQL 200.000 2 74 VFSKLENGGF100.000 example of VFSKIENGGL (2121) 200.000 improved peptideenhance P10 3 50 YPGIEIESRL 80.000 4 31 EPCGFEATYL 80.000 5 18EPGSGVRIVV 10.000 HLA-Cw*0602 1 85 YEKDLIEAI 6.600 2 65 FEIEINGQL 6.6003 21 SGVRIVVEY 6.000 4 31 EPCGFEATY 3.300 5 61 GTGAGEIEI 3.000HLA-Cw*0702 1 31 EPCGFEATY 24.000 2 21 SGVRIVVEY 19.200 3 42 LASAVKEQY8.800 4 77 KLENGGFPY 4.000 5 49 QYPGIEIES 2.880HLA-Cw*0702 (10-mer peptides) 1 20 GSGVRIVVEY 38.400 2 30 CEPCGFEATY16.000 3 41 ELASAVKEQY 16.000 4 50 YPGIEIESRL 7.920 5 76 SKLENGGFPY4.000

TABLE 5 Predicted C35 HLA Class I epitopes* HLA restriction elementInclusive amino acids Sequence A*0201   9-17  of SEQ ID NO: 2 SVAPPPEEVA*0201  10-17  of SEQ ID NO: 2 VAPPPEEV A*0201  16-23  of SEQ ID NO: 2EVEPGSGV A*0201  16-25  of SEQ ID NO: 2 EVEPGSGVRI A*0201 36-43  of SEQ ID NO: 2 EATYLELA A*0201  37-45  of SEQ ID NO: 2ATYLELASA A*0201  37-46  of SEQ ID NO: 2 ATYLELASAV A*0201 39-46  of SEQ ID NO: 2 YLELASAV A*0201  44-53  of SEQ ID NO: 2SAVKEQYPGI A*0201  45-53  of SEQ ID NO: 2 AVKEQYPGI A*0201 52-59  of SEQ ID NO: 2 GIEIESRL A*0201  54-62  of SEQ ID NO: 2EIESRLGGT A*0201  58-67  of SEQ ID NO: 2 RLGGTGAFEI A*0201 61-69  of SEQ ID NO: 2 GTGAFEIEI A*0201  66-73  of SEQ ID NO: 2EIEINGQL A*0201  66-74  of SEQ ID NO: 2 EIEINGQLV A*0201 88-96  of SEQ ID NO: 2 DLIEAIRRA A*0201  89-96  of SEQ ID NO: 2LIEAIRRA A*0201  92-101 of SEQ ID NO: 2 AIRRASNGET A*0201 95-102 of SEQ ID NO: 2 RASNGETL A*0201 104-113 of SEQ ID NO: 2KITNSRPPCV A*0201 105-113 of SEQ ID NO: 2 ITNSRPPCV A*0201105-114 of SEQ ID NO: 2 ITNSRPPCVI A*3101  16-24  of SEQ ID NO: 2EVEPGSGVR B*3501  30-38  of SEQ ID NO: 2 EPCGFEATY A*30101 96-104 of SEQ ID NO: 2 ASNGETLEK supermotif *predicted using rulesfound at the SYFPEITHI website(wysiwyg://35/http://134.2.96.221/scripts/hlaserver.dll/EpPredict.htm)and are based on the book “MHC Ligands and Peptide Motifs” by Rammensee,H. G., Bachmann, J. and S. Stevanovic. Chapman & Hall, New York, 1997.

TABLE 6 Predicted C35 HLA class II epitopes* Inclusive RestrictionSequence amino acids elements SGVRIVVEYCEPCGF 21-35 DRB1*0101(amino acids 21-35 of DRB1*0102 SEQ ID NO: 2) DRB1*0301 DRB1*0401DRB1*0404 DRB1*0405 DRB1*0410 DRB1*0421 DRB1*0701 DRB1*0801 DRB1*0804DRB1*0806 DRB1*1101 DRB1*1104 DRB1*1106 DRB1*1107 DRB1*1305 DRB1*1307DRB1*1321 DRB1*1501 DRB1*1502 DRB5*0101 SRLGGTGAFEIEINGQLVF 57-75DRB1*0101 (amino acids 57-75 of DRB1*0102 SEQ ID NO: 2) DRB1*0301DRB1*0401 DRB1*0402 DRB1*0421 DRB1*0701 DRB1*0804 DRB1*0806 DRB1*1101DRB1*1104 DRB1*1106 DRB1*1305 DRB1*1321 DRB1*1501 DRB1*1502 DRB5*0101GAFEIEINGQLVFSKLENGGF 63-83 DRB1*0101 (amino acids 63-83 of DRB1*0102SEQ ID NO: 2) DRB1*0301 DRB1*0401 DRB1*0402 DRB1*0404 DRB1*0405DRB1*0410 DRB1*0421 DRB1*0701 DRB1*0804 DRB1*0806 DRB1*1101 DRB1*1104DRB1*1106 DRB1*1107 DRB1*1305 DRB1*1307 DRB1*1311 DRB1*1321 DRB1*1501DRB1*1502 DRB5*0101 FPYEKDLIEAIRRASNGETLE  83-103 DRB1*0101(amino acids 83-103 of DRB1*0102 SEQ ID NO: 2) DRB1*0301 DRB1*0401DRB1*0402 DRB1*0404 DRB1*0405 DRB1*0410 DRB1*0421 DRB1*0701 DRB1*0801DRB1*0802 DRB1*0804 DRB1*0806 DRB1*1101 DRB1*1104 DRB1*1106 DRB1*1107DRB1*1305 DRB1*1307 DRB1*1311 DRB1*1321 DRB1*1501 DRB1*1502 DRB5*0101*Class II MHC epitopes predicted using TEPITOPE software. Sturniolo, T.,et al. 1999. Generation of tissue specific and promiscuous HLA liganddatabases using DNA microarrays and virtual HLA class II matrices.Nature Biotechnology 17:555571.

In the present invention, “epitopes” refer to C35 polypeptide fragmentshaving antigenic or immunogenic activity in an animal, especially in ahuman, or that are capable of eliciting a T lymphocyte response in ananimal, preferably a human. A preferred embodiment of the presentinvention relates to a C35 polypeptide fragment comprising a C35 peptideepitope, as well as the polynucleotide encoding this fragment. A furtherpreferred embodiment of the present invention relates to a C35polypeptide fragment consisting of an epitope, as well as thepolynucleotide encoding this fragment. In specific preferred embodimentsof the present invention, the epitope comprises a C35 fragment listed inany of Tables 1-3 or 5-6, exclusive of E-100 to R-109 of SEQ ID NO:2. Inanother preferred embodiment of the present invention, the epitopeconsists of a C35 fragment listed in any of Tables 1-3 and 5-6 exclusiveof E-100 to R-109 of SEQ ID NO:2. A region of a protein molecule towhich an antibody can bind is defined as an “antigenic epitope.” Incontrast, an “immunogenic epitope” is defined as a part of a proteinthat elicits T cell response. (See, for instance, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998 4002 (1983)). Thus, a further preferredembodiment of the present invention is an immunogenic C35 peptidefragment that is capable of eliciting a T cell response when bound tothe peptide binding cleft of an MHC molecule. In a specific preferredembodiment, the immunogenic C35 peptide fragment comprises an epitopelisted in any of Tables 1-3 or 5-6 exclusive of E-100 to R-109 of SEQ IDNO:2. In another preferred embodiment, the immunogenic C35 peptidefragment consists of an epitope listed in any of Tables 1-3 or 5-6exclusive of E-100 to R-109 of SEQ ID NO:2. Further embodiments of theinvention are directed to pharmaceutical formulations and vaccinecompositions comprising said immunogenic C35 peptide fragments or thepolynucleotides encoding them.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA82:51315135 (1985) further described in U.S. Pat. No. 4,631,211.)

The sequence of peptide epitopes known to bind to specific MHC moleculescan be modified at the known peptide anchor positions in predictableways that act to increase MHC binding affinity. Such “epitopeenhancement” has been employed to improve the immunogenicity of a numberof different MHC class I or MHC class II binding peptide epitopes(Berzofsky, J. A. et al., Immunol. Rev. 170:151-72 (1999); Ahlers, J. D.et al., Proc. Natl. Acad. Sci U.S.A. 94:10856-61 (1997); Overwijk, etal., J. Exp. Med. 188:277-86 (1998); Parkhurst, M. R. et al., J.Immunol. 157:2539-48 (1996)). Accordingly, a further embodiment of theinvention is directed to such enhanced C35 peptide epitope analogs, andto the polynucleotides encoding such analogs.

In the present invention, antigenic epitopes preferably contain asequence of at least seven, more preferably at least nine, and mostpreferably between about 15 to about 30 amino acids. Antigenic epitopesare useful to raise antibodies, including monoclonal antibodies, thatspecifically bind the epitope. (See, for instance, Wilson et al., Cell37:767778 (1984); Sutcliffe, J. G. et al., Science 219:660666 (1983).)

Similarly, immunogenic epitopes can be used to induce B cells and Tcells according to methods well known in the art. (See Sutcliffe et al.,supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA82:910914; and Bittle, F. J. et al., J. Gen. Virol. 66:23472354 (1985).)The immunogenic epitopes may be presented together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse) or, if it is long enough (at least about 25 amino acids), withouta carrier. However, immunogenic epitopes comprising as few as 9 aminoacids have been shown to be sufficient to raise antibodies capable ofbinding to, at the very least, linear epitopes in a denaturedpolypeptide (e.g., in Western blotting.)

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to protein. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less nonspecific tissue binding than an intact antibody.(Wahl et al., J. Nucl. Med. 24:316325 (1983).) Thus, for someapplications these fragments are preferred, as well as the products of aFab or other immunoglobulin expression library. Moreover, antibodies ofthe present invention include chimeric, single chain, and humanizedantibodies.

Diagnostic and Therapeutic Uses of Antibodies

The present invention further relates to C35 antibodies, C35 antibodyfragments and antibody conjugates and singlechain immunotoxins reactivewith human carcinoma cells, particularly human breast and bladdercarcinoma cells.

Table 7 provides a list of C35-specific monoclonal antibodies that havebeen isolated and characterized for use in different applications.

TABLE 7 C35 -Specific Murine Monoclonal Antibodies Western FlowImmunohisto- Fusion Hybridoma ELISA Isotype Blot Cytometry chemistryalpha 1F5 positive IgM positive positive 1F7 positive IgM positive 1F11positive IgM positive 2D9 positive IgM positive positive positive beta2G3 positive IgG1 2G8 positive 2G10 positive IgG3 2G11 positive IgG3 3F9positive IgG1 4D11 positive IgG1 4G3 positive IgG3 7C2 positive IgM 8B11positive IgM 8G2 positive IgM 10F4 positive IgG1 11B10 positive IgMpositive 12B10 positive 16C10 positive IgM 16F10 positive ELISA assay onbacterially-synthesized C35 blank = not determined

As used in this example, the following words or phrases have themeanings specified.

As used in this example, “joined” means to couple directly or indirectlyone molecule with another by whatever means, e.g., by covalent bonding,by noncovalent bonding, by ionic bonding, or by nonionic bonding.Covalent bonding includes bonding by various linkers such as thioetherlinkers or thioester linkers. Direct coupling involves one moleculeattached to the molecule of interest. Indirect coupling involves onemolecule attached to another molecule not of interest which in turn isattached directly or indirectly to the molecule of interest.

As used in this example, “recombinant molecule” means a moleculeproduced by genetic engineering methods.

As used in this example, “fragment” is defined as at least a portion ofthe variable region of the immunoglobulin molecule which binds to itstarget, i.e. the antigen binding region. Some of the constant region ofthe immunoglobulin may be included.

As used in this example, an “immunoconjugate” means any molecule orligand such as an antibody or growth factor chemically or biologicallylinked to a cytotoxin, a radioactive agent, an antitumor drug or atherapeutic agent. The antibody or growth factor may be linked to thecytotoxin, radioactive agent, antitumor drug or therapeutic agent at anylocation along the molecule so long as it is able to bind its target.Examples of immunoconjugates include immunotoxins and antibodyconjugates.

As used in this example, “selectively killing” means killing those cellsto which the antibody binds.

As used in this example, examples of “carcinomas” include bladder,breast, colon, liver, lung, ovarian, and pancreatic carcinomas.

As used in this example, “immunotoxin” means an antibody or growthfactor chemically or biologically linked to a cytotoxin or cytotoxicagent.

As used in this example, an “effective amount” is an amount of theantibody, immunoconjugate, recombinant molecule which kills cells orinhibits the proliferation thereof.

As used in this example, “competitively inhibits” means being capable ofbinding to the same target as another molecule. With regard to anantibody, competitively inhibits mean that the antibody is capable ofrecognizing and binding the same antigen binding region to which anotherantibody is directed.

As used in this example, “antigenbinding region” means that part of theantibody, recombinant molecule, the fusion protein, or theimmunoconjugate of the invention which recognizes the target or portionsthereof.

As used in this example, “therapeutic agent” means any agent useful fortherapy including antitumor drugs, cytotoxins, cytotoxin agents, andradioactive agents.

As used in this example, “antitumor drug” means any agent useful tocombat cancer including, but not limited to, cytotoxins and agents suchas antimetabolites, alkylating agents, anthracyclines, antibiotics,antimitotic agents, procarbazine, hydroxyurea, asparaginase,corticosteroids, mytotane (O,P′(DDD)), interferons and radioactiveagents.

As used in this example, “a cytotoxin or cytotoxic agent” means anyagent that is detrimental to cells. Examples include taxol, cytochalasinB, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

As used in this example, “radioisotope” includes any radioisotope whichis effective in destroying a tumor. Examples include, but are notlimited to, cobalt60 and Xrays. Additionally, naturally occurringradioactive elements such as uranium, radium, and thorium whichtypically represent mixtures of radioisotopes, are suitable examples ofa radioactive agent.

As used in this example, “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular or subcutaneous administration, or theimplantation of a slowrelease device such as a miniosmotic pump, to thesubject.

As used in this example, “directly” means the use of antibodies coupledto a label. The specimen is incubated with the labeled antibody, unboundantibody is removed by washing, and the specimen may be examined.

As used in this example, “indirectly” means incubating the specimen withan unconjugated antibody, washing and incubating with afluorochromeconjugated antibody. The second or “sandwich” antibody thusreveals the presence of the first.

As used in this example “reacting” means to recognize and bind thetarget. The binding may be nonspecific. Specific binding is preferred.

As used in this example, “curing” means to provide substantiallycomplete tumor regression so that the tumor is not palpable for a periodof time, i.e., >/=10 tumor volume doubling delays (TVDD=the time in daysthat it takes for control tumors to double in size).

As used in this example, “tumor targeted antibody” means any antibodywhich recognizes the C35 antigen on tumor (i.e., cancer) cells.

As used in this example, “inhibit proliferation” means to interfere withcell growth by whatever means.

As used in this example, “mammalian tumor cells” include cells fromanimals such as human, ovine, porcine, murine, bovine animals.

As used in this example, “pharmaceutically acceptable carrier” includesany material which when combined with the antibody retains theantibody's immunogenicity and is nonreactive with the subject's immunesystems. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Other carriers may also include sterile solutions,tablets including coated tablets and capsules.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, magnesiumor calcium stearate, talc, vegetable fats or oils, gums, glycols, orother known excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well known conventional methods.

The present invention relates to C35 antibodies that are highly specificfor carcinoma cells. More particularly, the antibodies react with arange of carcinomas such as breast, bladder, lung, ovary and coloncarcinomas, while showing none or limited reactivity with normal humantissues or other types of tumors such as, for example, sarcomas orlymphomas.

The term “C35 antibody” as used herein includes whole, intact polyclonaland monoclonal antibody materials, and chimeric antibody molecules. TheC35 antibody described above includes any fragments thereof containingthe active antigenbinding region of the antibody such as Fab, F(ab′)2and Fv fragments, using techniques well established in the art [see,e.g., Rousseaux et al., “Optimal Conditions For The Preparation ofProteolytic Fragments From Monoclonal IgG of Different Rat IgGSubclasses”, in Methods Enzymol., 121:66369 (Academic Press 1986)]. TheC35 antibody of the invention also includes fusion proteins.

Also included within the scope of the invention are antiidiotypicantibodies to the C35 antibody of the invention. These antiidiotypicantibodies can be produced using the C35 antibody and/or the fragmentsthereof as immunogen and are useful for diagnostic purposes in detectinghumoral response to tumors and in therapeutic applications, e.g., in avaccine, to induce an antitumor response in patients [see, e.g., Nepomet al., “AntiIdiotypic Antibodies And The Induction Of Specific TumorImmunity”, in Cancer And Metastasis Reviews, 6:487501 (1987)].

In addition, the present invention encompasses antibodies that arecapable of binding to the same antigenic determinant as the C35antibodies and competing with the antibodies for binding at that site.These include antibodies having the same antigenic specificity as theC35 antibodies but differing in species origin, isotype, bindingaffinity or biological functions (e.g., cytotoxicity). For example,class, isotype and other variants of the antibodies of the inventionhaving the antigenbinding region of the C35 antibody can be constructedusing recombinant classswitching and fusion techniques known in the art[see, e.g., Thammana et al., “Immunoglobulin Heavy Chain Class SwitchFrom IgM to IgG In A Hybridoma”, Eur. J. Immunol., 13:614 (1983); Spiraet al., “The Identification of Monoclonal Class Switch Variants bySubselection and ELISA Assay”, J. Immunol. Meth. 74:30715 (1984);Neuberger et al., “Recombinant Antibodies Possessing Novel EffectorFunctions”, Nature 312: 614608 (1984); and Oi et al., “ChimericAntibodies”, Biotechniques 4 (3):21421 (1986)]. Thus, other chimericantibodies or other recombinant antibodies (e.g., fusion proteinswherein the antibody is combined with a second protein such as alymphokine or a tumor inhibitory growth factor) having the same bindingspecificity as the C35-specific antibodies fall within the scope of thisinvention.

Genetic engineering techniques known in the art may be used as describedherein to prepare recombinant immunotoxins produced by fusing antigenbinding regions of antibody C35 to a therapeutic or cytotoxic agent atthe DNA level and producing the cytotoxic molecule as a chimericprotein. Examples of therapeutic agents include, but are not limited to,antimetabolites, alkylating agents, anthracyclines, antibiotics, andantimitotic agents. Antimetabolites include methotrexate,6mercaptopurine, 6thioguanine, cytarabine, 5-fluorouracil decarbazine.Alkylating agents include mechlorethamine, thioepa chlorambucil,melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide,busulfan, dibromomannitol, streptozotocin, mitomycin C, andcisdichlorodiamine platinum (II) (DDP) cisplatin. Anthracyclines includedaunorubicin (formerly daunomycin) and doxorubicin (also referred toherein as adriamycin). Additional examples include mitozantrone andbisantrene. Antibiotics include dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC). Antimytotic agentsinclude vincristine and vinblastine (which are commonly referred to asvinca alkaloids). Other cytotoxic agents include procarbazine,hydroxyurea, asparaginase, corticosteroids, mytotane (O,P′(DDD)),interferons. Further examples of cytotoxic agents include, but are notlimited to, ricin, doxorubicin, taxol, cytochalasin B, gramicidin D,ethidium bromide, etoposide, tenoposide, colchicin, dihydroxy anthracindione, 1dehydrotestosterone, and glucocorticoid.

Clearly analogs and homologs of such therapeutic and cytotoxic agentsare encompassed by the present invention. For example, thechemotherapuetic agent aminopterin has a correlative improved analognamely methotrexate. Further, the improved analog of doxorubicin is anFechelate. Also, the improved analog for 1methylnitrosourea islomustine. Further, the improved analog of vinblastine is vincristine.Also, the improved analog of mechlorethamine is cyclophosphamide.

Recombinant immunotoxins, particularly singlechain immunotoxins, have anadvantage over drug/antibody conjugates in that they are more readilyproduced than these conjugates, and generate a population of homogenousmolecules, i.e. single peptides composed of the same amino acidresidues. The techniques for cloning and expressing DNA sequencesencoding the amino acid sequences corresponding to C35 singlechainimmunotoxins, e.g synthesis of oligonucleotides, PCR, transformingcells, constructing vectors, expression systems, and the like are wellestablished in the art, and most practitioners are familiar with thestandard resource materials for specific conditions and procedures [see,e.g., Sambrook et al., eds., Molecular Cloning, A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory Press (1989)].

The following include preferred embodiments of the immunoconjugates ofthe invention. Other embodiments which are known in the art areencompassed by the invention. The invention is not limited to thesespecific immunoconjugates, but also includes other immunoconjugatesincorporating antibodies and/or antibody fragments according to thepresent invention.

The conjugates comprise at least one drug molecule connected by a linkerof the invention to a targeting ligand molecule that is reactive withthe desired target cell population. The ligand molecule can be animmunoreactive protein such as an antibody, or fragment thereof, anonimmunoreactive protein or peptide ligand such as bombesin or, abinding ligand recognizing a cell associated receptor such as a lectinor steroid molecule.

Further, because the conjugates of the invention can be used formodifying a given biological response, the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha interferon, betainterferon, nerve growth factor, platelet derived growth factor, tissueplasminogen activator; or, biological response modifiers such as, forexample, lymphokines, interleukin1 (“IL1”), interleukin2 (“IL2”),interleukin6 (“IL6”), granulocyte macrophase colony stimulating factor(“GMCSF”), granulocyte colony stimulating factor (“GCSF”), or othergrowth factors.

The preferred drugs for use in the present invention are cytotoxicdrugs, particularly those which are used for cancer therapy. Such drugsinclude, in general, alkylating agents, antiproliferative agents,tubulin binding agents and the like. Preferred classes of cytotoxicagents include, for example, the anthracycline family of drugs, thevinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides,the pteridine family of drugs, diynenes, and the podophyllotoxins.Particularly useful members of those classes include, for example,adriamycin, caminomycin, daunorubicin, aminopterin, methotrexate,methopterin, dichloromethotrexate, mitomycin C, porfiromycin,5fluorouracil, 6mercaptopurine, cytosine arabinoside, podophyllotoxin,or podophyllotoxin derivatives such as etoposide or etoposide phosphate,melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosineand the like. As noted previously, one skilled in the art may makechemical modifications to the desired compound in order to makereactions of that compound more convenient for purposes of preparingconjugates of the invention.

As noted, one skilled in the art will appreciate that the invention alsoencompasses the use of antigen recognizing immunoglobulin fragments.Such immunoglobulin fragments may include, for example, the Fab′,F(ab′)2, F[v] or Fab fragments, or other antigen recognizingimmunoglobulin fragments. Such immunoglobulin fragments can be prepared,for example, by proteolytic enzyme digestion, for example, by pepsin orpapain digestion, reductive alkylation, or recombinant techniques. Thematerials and methods for preparing such immunoglobulin fragments arewellknown to those skilled in the art. See generally, Parham, J.Immunology 131:2895 (1983); Lamoyi et al., J. Immunological Methods56:235 (1983); Parham, id., 53, 133 (1982); and Matthew et al., id., 50,239 (1982).

The immunoglobulin can be a “chimeric antibody” as that term isrecognized in the art. Also, the immunoglobulin may be a “bifunctional”or “hybrid” antibody, that is, an antibody which may have one arm havinga specificity for one antigenic site, such as a tumor associated antigenwhile the other arm recognizes a different target, for example, a haptenwhich is, or to which is bound, an agent lethal to the antigenbearingtumor cell. Alternatively, the bifunctional antibody may be one in whicheach arm has specificity for a different epitope of a tumor associatedantigen of the cell to be therapeutically or biologically modified. Inany case, the hybrid antibodies have a dual specificity, preferably withone or more binding sites specific for the hapten of choice or one ormore binding sites specific for a target antigen, for example, anantigen associated with a tumor, an infectious organism, or otherdisease state.

Biological bifunctional antibodies are described, for example, inEuropean Patent Publication, EPA 0 105 360, to which those skilled inthe art are referred. Such hybrid or bifunctional antibodies may bederived, as noted, either biologically, by cell fusion techniques, orchemically, especially with crosslinking agents or disulfidebridgeforming reagents, and may be comprised of whose antibodies and/orfragments thereof. Methods for obtaining such hybrid antibodies aredisclosed, for example, in PCT application WO83/03679, published Oct.27, 1983, and published European Application EPA 0 217 577, publishedApr. 8, 1987. Particularly preferred bifunctional antibodies are thosebiologically prepared from a “polydome” or “quadroma” or which aresynthetically prepared with crosslinking agents such asbis(maleimideo)methyl ether (“BMME”), or with other crosslinking agentsfamiliar to those skilled in the art.

In addition the immunoglobulin may be a single chain antibody (“SCA”).These may consist of single chain Fv fragments (“scFv”) in which thevariable light (“V[L]”) and variable heavy (“V[H]”) domains are linkedby a peptide bridge or by disulfide bonds. Also, the immunoglobulin mayconsist of single V[H] domains (dAbs) which possess antigenbindingactivity. See, e.g., G. Winter and C. Milstein, Nature 349:295 (1991);R. Glockshuber et al., Biochemistry 29:1362 (1990); and, E. S. Ward etal., Nature 341: 544 (1989).

Especially preferred for use in the present invention are chimericmonoclonal antibodies, preferably those chimeric antibodies havingspecificity toward a tumor associated antigen. As used in this example,the term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e. binding region, from one source or species andat least a portion of a constant region derived from a different sourceor species, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are preferred in certain applications of the invention,particularly human therapy, because such antibodies are readily preparedand may be less immunogenic than purely murine monoclonal antibodies.Such murine/human chimeric antibodies are the product of expressedimmunoglobulin genes comprising DNA segments encoding murineimmunoglobulin variable regions and DNA segments encoding humanimmunoglobulin constant regions. Other forms of chimeric antibodiesencompassed by the invention are those in which the class or subclasshas been modified or changed from that of the original antibody. Such“chimeric” antibodies are also referred to as “classswitchedantibodies”. Methods for producing chimeric antibodies involveconventional recombinant DNA and gene transfection techniques now wellknown in the art. See, e.g., Morrison, S. L. et al., Proc. Nat'l Acad.Sci. 81:6851 (1984).

Encompassed by the term “chimeric antibody” is the concept of “humanizedantibody”, that is those antibodies in which the framework or“complementarity” determining regions (“CDR”) have been modified tocomprise the CDR of an immunoglobulin of different specificity ascompared to that of the parent immunoglobulin. In a preferredembodiment, a murine CDR is grafted into the framework region of a humanantibody to prepare the “humanized antibody”. See, e.g., L. Riechmann etal., Nature 332:323 (1988); M. S. Neuberger et al., Nature 314:268(1985). Particularly preferred CDR'S correspond to those representingsequences recognizing the antigens noted above for the chimeric andbifunctional antibodies. The reader is referred to the teaching of EPA 0239 400 (published Sep. 30, 1987), for its teaching of CDR modifiedantibodies.

One skilled in the art will recognize that a bifunctionalchimericantibody can be prepared which would have the benefits of lowerimmunogenicity of the chimeric or humanized antibody, as well as theflexibility, especially for therapeutic treatment, of the bifunctionalantibodies described above. Such bifunctionalchimeric antibodies can besynthesized, for instance, by chemical synthesis using crosslinkingagents and/or recombinant methods of the type described above. In anyevent, the present invention should not be construed as limited in scopeby any particular method of production of an antibody whetherbifunctional, chimeric, bifunctionalchimeric, humanized, or anantigen-recognizing fragment or derivative thereof.

In addition, the invention encompasses within its scope immunoglobulins(as defined above) or immunoglobulin fragments to which are fused activeproteins, for example, an enzyme of the type disclosed in Neuberger, etal., PCT application, WO86/01533, published Mar. 13, 1986. Thedisclosure of such products is incorporated herein by reference.

As noted, “bifunctional”, “fused”, “chimeric” (including humanized), and“bifunctionalchimeric” (including humanized) antibody constructions alsoinclude, within their individual contexts constructions comprisingantigen recognizing fragments. As one skilled in the art will recognize,such fragments could be prepared by traditional enzymatic cleavage ofintact bifunctional, chimeric, humanized, or chimericbifunctionalantibodies. If, however, intact antibodies are not susceptible to suchcleavage, because of the nature of the construction involved, the notedconstructions can be prepared with immunoglobulin fragments used as thestarting materials; or, if recombinant techniques are used, the DNAsequences, themselves, can be tailored to encode the desired “fragment”which, when expressed, can be combined in vivo or in vitro, by chemicalor biological means, to prepare the final desired intact immunoglobulin“fragment”. It is in this context, therefore, that the term “fragment”is used.

Furthermore, as noted above, the immunoglobulin (antibody), or fragmentthereof, used in the present invention may be polyclonal or monoclonalin nature. Monoclonal antibodies are the preferred immunoglobulins,however. The preparation of such polyclonal or monoclonal antibodies nowis well known to those skilled in the art who, of course, are fullycapable of producing useful immunoglobulins which can be used in theinvention. See, e.g., G. Kohler and C. Milstein, Nature 256: 495 (1975).In addition, hybridomes and/or monoclonal antibodies which are producedby such hybridomas and which are useful in the practice of the presentinvention are publicly available from sources such as the American TypeCulture Collection (“ATCC”) 10801 University Blvd., Manassas, Va. 20110.

Particularly preferred monoclonal antibodies for use in the presentinvention are those which recognize tumor associated antigens.

Diagnostic Techniques

Serologic diagnostic techniques involve the detection and quantitiationof tumorassociated antigens that have been secreted or “shed” into theserum or other biological fluids of patients thought to be sufferingfrom carcinoma. Such antigens can be detected in the body fluids usingtechniques known in the art such as radioimmunoassays (RIA) orenzymelinked immunosorbent assays (ELISA) wherein an antibody reactivewith the “shed” antigen is used to detect the presence of the antigen ina fluid sample [see, e.g., Uotila et al., “TwoSite Sandwich ELISA WithMonoclonal Antibodies To Human AFP”, J. Immunol. Methods 42:11 (1981)and Allum et al., supra at pp. 4851]. These assays, using the C35antibodies disclosed herein, can therefore be used for the detection inbiological fluids of the antigen with which the C35 antibodies react andthus the detection of human carcinoma in patients. Thus, it is apparentfrom the foregoing that the C35 antibodies of the invention can be usedin most assays involving antigenantibody reactions. These assaysinclude, but are not limited to, standard RIA techniques, both liquidand solid phase, as well as ELISA assays, ELISPOT, immunofluorescencetechniques, and other immunocytochemical assays [see, e.g., Sikora etal. (eds.), Monoclonal Antibodies, pp. 3252 (Blackwell ScientificPublications 1984)].

The invention also encompasses diagnostic kits for carrying out theassays described above. In one embodiment, the diagnostic kit comprisesthe C35 monoclonal antibody, fragments thereof, fusion proteins orchimeric antibody of the invention, and a conjugate comprising aspecific binding partner for the C35 antibody and a label capable ofproducing a detectable signal. The reagents can also include ancillaryagents such as buffering agents and protein stabilizing agents (e.g.,polysaccharides). The diagnostic kit can further comprise, wherenecessary, other components of the signalproducing system includingagents for reducing background interference, control reagents or anapparatus or container for conducting the test.

In another embodiment, the diagnostic kit comprises a conjugate of theC35 antibodies of the invention and a label capable of producing adetectable single. Ancillary agents as mentioned above can also bepresent.

The C35 antibody of the invention is also useful for in vivo diagnosticapplications for the detection of human carcinomas. One such approachinvolves the detection of tumors in vivo by tumor imaging techniques.According to this approach, the C35 antibody is labeled with anappropriate imaging reagent that produces a detectable signal. Examplesof imaging reagents that can be used include, but at not limited to,radiolabels such as <131>I, <111>In, <123>I, <99m>Tc, <32>P, <125>I,<3>H, and <14>C, fluorescent labels such as fluorescein and rhodamine,and chemiluninescers such as luciferin. The antibody can be labeled withsuch reagents using techniques known in the art. For example, see Wenseland Meares, Radioimmunoimaging And Radioimmunotherapy, Elsevier, NewYork (1983) for techniques relating to the radiolabeling of antibodies[see also, Colcher et al., “Use of Monoclonal Antibodies asRadiopharmaceuticals for the Localization of Human Carcinoma Xenograftsin Athymic Mice”, Meth. Enzymol. 121:80216 (1986)].

In the case of radiolabeled antibody, the antibody is administered tothe patient, localizes to the tumor bearing the antigen with which theantibody reacts, and is detected or “imaged” in vivo using knowntechniques such as radionuclear scanning using, e.g., a gamma camera oremission tomography [see, e.g., Bradwell et al., “Developments InAntibody Imaging”, in Monoclonal Antibodies for Cancer Detection andTherapy, Baldwin, et al. (eds.), pp. 6585 (Academic Press 1985)]. theantibody is administered to the patient in a pharmaceutically acceptablecarrier such as water, saline, Ringer's solution, Hank's solution ornonaqueous carriers such as fixed oils. The carrier may also containsubstances that enhance isotonicity and chemical stability of theantibody such as buffers or preservatives. The antibody formulation isadministered, for example, intravenously, at a dosage sufficient toprovide enough gamma emission to allow visualization of the tumor targetsite. Sufficient time should be allowed between administration of theantibody and detection to allow for localization to the tumor target.For a general discussion of tumor imaging, see Allum et al, supra, atpp. 5155.

Therapeutic Applications of C35 Antibodies

The properties of the C35 antibody suggest a number of in vivotherapeutic applications.

First, the C35 antibody can be used alone to target and kill tumor cellsin vivo. The antibody can also be used in conjunction with anappropriate therapeutic agent to treat human carcinoma. For example, theantibody can be used in combination with standard or conventionaltreatment methods such as chemotherapy, radiation therapy or can beconjugated or linked to a therapeutic drug, or toxin, as well as to alymphokine or a tumorinhibitory growth factor, for delivery of thetherapeutic agent to the site of the carcinoma.

Techniques for conjugating such therapeutic agents to antibodies arewell known [see, e.g., Arnon et al., “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy”, in Monoclonal Antibodiesand Cancer Therapy, Reisfeld et al. (eds.), pp. 24356 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 62353(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475506(1985); and Thorpe et al., “The Preparation And Cytotoxic Properties OfAntibodyToxin Conjugates”, Immunol. Rev., 62:11958 (1982)].

Alternatively, the C35 antibody can be coupled to highenergy radiation,e.g., a radioisotope such as <131>I, which, when localized at the tumorsite, results in a killing of several cell diameters [see, e.g., Order,“Analysis, Results, and Future Prospective of The Therapeutic Use ofRadiolabeled Antibody in Cancer Therapy”, in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al. (eds.), pp. 30316 (AcademicPress 1985)]. According to yet another embodiment, the C35 antibody canbe conjugated to a second antibody to form an antibody heteroconjugatefor the treatment of tumor cells as described by Segal in U.S. Pat. No.4,676,980.

Still other therapeutic applications for the C35 antibody of theinvention include conjugation or linkage, e.g., by recombinant DNAtechniques, to an enzyme capable of converting a prodrug into acytotoxic drug and the use of that antibodyenzyme conjugate incombination with the prodrug to convert the prodrug to a cytotoxic agentat the tumor site [see, e.g., Senter et al., “AntiTumor Effects ofAntibodyalkaline Phosphatase”, Proc. Natl. Acad. Sci. USA 85:484246(1988); “Enhancement of the in vitro and in vivo Antitumor Activites ofPhosphorylated Mitocycin C and Etoposide Derivatives by MonoclonalAntibodyAlkaline Phosphatase Conjugates”, Cancer Research 49:57895792(1989); and Senter, “Activation of Prodrugs by AntibodyEnzymeConjugates: A New Approach to Cancer Therapy,” FASEB J. 4:188193(1990)].

Still another therapeutic use for the C35 antibody involves use, eitherin the presence of complement or as part of an antibodydrug orantibodytoxin conjugate, to remove tumor cells from the bone marrow ofcancer patients. According to this approach, autologous bone marrow maybe purged ex vivo by treatment with the antibody and the marrow infusedback into the patient [see, e.g., Ramsay et al., “Bone Marrow PurgingUsing Monoclonal Antibodies”, J. Clin. Immunol., 8(2):8188 (1988)].

Furthermore, chimeric C35, recombinant immunotoxins and otherrecombinant constructs of the invention containing the specificity ofthe antigenbinding region of the C35 monoclonal antibody, as describedearlier, may be used therapeutically. For example, the singlechainimmunotoxins of the invention, may be used to treat human carcinoma invivo.

Similarly, a fusion protein comprising at least the antigenbindingregion of the C35 antibody joined to at least a functionally activeportion of a second protein having antitumor activity, e.g., alymphokine or oncostatin can be used to treat human carcinoma in vivo.Furthermore, recombinant techniques known in the art can be used toconstruct bispecific antibodies wherein one of the binding specificitiesof the antibody is that of C35, while the other binding specificity ofthe antibody is that of a molecule other than C35.

Finally, antiidiotypic antibodies of the C35 antibody may be usedtherapeutically in active tumor immunization and tumor therapy [see,e.g., Hellstrom et al., “Immunological Approaches To Tumor Therapy:Monoclonal Antibodies, Tumor Vaccines, And Antiudiotypes”, in CovalentlyModified Antigens And Antibodies In Diagnosis And Therapy, supra at pp.3541].

The present invention provides a method for selectively killing tumorcells expressing the antigen that specifically binds to the C35monoclonal antibody or functional equivalent. This method comprisesreacting the immunoconjugate (e.g. the immunotoxin) of the inventionwith said tumor cells. These tumor cells may be from a human carcinoma.

Additionally, this invention provides a method of treating carcinomas(for example human carcinomas) in vivo. This method comprisesadministering to a subject a pharmaceutically effective amount of acomposition containing at least one of the immunoconjugates (e.g. theimmunotoxin) of the invention.

In accordance with the practice of this invention, the subject may be ahuman, equine, porcine, bovine, murine, canine, feline, and aviansubjects. Other warm blooded animals are also included in thisinvention.

The present invention also provides a method for curing a subjectsuffering from a cancer. The subject may be a human, dog, cat, mouse,rat, rabbit, horse, goat, sheep, cow, chicken. The cancer may beidentified as a breast, bladder, retinoblastoma, papillarycystadenocarcinoma of the ovary, Wilm's tumor, or small cell lungcarcinoma and is generally characterized as a group of cells havingtumor associated antigens on the cell surface. This method comprisesadministering to the subject a cancer killing amount of a tumor targetedantibody joined to a cytotoxic agent. Generally, the joining of thetumor targeted antibody with the cytotoxic agent is made underconditions which permit the antibody so joined to bind its target on thecell surface. By binding its target, the tumor targeted antibody actsdirectly or indirectly to cause or contribute to the killing of thecells so bound thereby curing the subject.

Also provided is a method of inhibiting the proliferation of mammaliantumor cells which comprises contacting the mammalian tumor cells with asufficient concentration of the immunoconjugate of the invention so asto inhibit proliferation of the mammalian tumor cells.

The subject invention further provides methods for inhibiting the growthof human tumor cells, treating a tumor in a subject, and treating aproliferative type disease in a subject. These methods compriseadministering to the subject an effective amount of the composition ofthe invention.

It is apparent therefore that the present invention encompassespharmaceutical compositions, combinations and methods for treating humancarcinomas. For example, the invention includes pharmaceuticalcompositions for use in the treatment of human carcinomas comprising apharmaceutically effective amount of a C35 antibody and apharmaceutically acceptable carrier.

The compositions may contain the C35 antibody or antibody fragments,either unmodified, conjugated to a therapeutic agent (e.g., drug, toxin,enzyme or second antibody) or in a recombinant form (e.g., chimeric C35,fragments of chimeric C35, bispecific C35 or singlechain immunotoxinC35). The compositions may additionally include other antibodies orconjugates for treating carcinomas (e.g., an antibody cocktail).

The antibody, antibody conjugate and immunotoxin compositions of theinvention can be administered using conventional modes of administrationincluding, but not limited to, intravenous, intraperitoneal, oral,intralymphatic or administration directly into the tumor. Intravenousadministration is preferred.

The compositions of the invention may be in a variety of dosage formswhich include, but are not limited to, liquid solutions or suspension,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The compositions of the invention also preferably include conventionalpharmaceutically acceptable carriers and adjuvants known in the art suchas human serum albumin, ion exchangers, alumina, lecithin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,and salts or electrolytes such as protamine sulfate.

The most effective mode of administration and dosage regimen for thecompositions of this invention depends upon the severity and course ofthe disease, the patient's health and response to treatment and thejudgment of the treating physician. Accordingly, the dosages of thecompositions should be titrated to the individual patient. Nevertheless,an effective dose of the compositions of this invention may be in therange of from about 1 to about 2000 mg/kg.

The molecules described herein may be in a variety of dosage forms whichinclude, but are not limited to, liquid solutions or suspensions,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The most effective mode of administration and dosage regimen for themolecules of the present invention depends upon the location of thetumor being treated, the severity and course of the cancer, thesubject's health and response to treatment and the judgment of thetreating physician. Accordingly, the dosages of the molecules should betitrated to the individual subject.

The interrelationship of dosages for animals of various sizes andspecies and humans based on mg/kg of surface area is described byFreireich, E. J., et al. Cancer Chemother., Rep. 50 (4): 219244 (1966).Adjustments in the dosage regimen may be made to optimize the tumor cellgrowth inhibiting and killing response, e.g., doses may be divided andadministered on a daily basis or the dose reduced proportionallydepending upon the situation (e.g., several divided doses may beadministered daily or proportionally reduced depending on the specifictherapeutic situation.

It would be clear that the dose of the composition of the inventionrequired to achieve cures may be further reduced with scheduleoptimization.

In accordance with the practice of the invention, the pharmaceuticalcarrier may be a lipid carrier. The lipid carrier may be a phospholipid.Further, the lipid carrier may be a fatty acid. Also, the lipid carriermay be a detergent. As used herein, a detergent is any substance thatalters the surface tension of a liquid, generally lowering it.

In one example of the invention, the detergent may be a nonionicdetergent. Examples of nonionic detergents include, but are not limitedto, polysorbate 80 (also known as Tween 80 or (polyoxyethylenesorbitanmonooleate), Brij, and Triton (for example Triton WR1339 and TritonA20).

Alternatively, the detergent may be an ionic detergent. An example of anionic detergent includes, but is not limited to, alkyltrimethylammoniumbromide.

Additionally, in accordance with the invention, the lipid carrier may bea liposome. As used in this application, a “liposome” is any membranebound vesicle which contains any molecules of the invention orcombinations thereof.

Vaccine Formulations

The C35 epitopes can be produced in quantity by recombinant DNA methodsand formulated with an adjuvant that promotes a cell-mediated immuneresponse. The present invention encompasses the expression of the C35polypeptides, or C35 epitopes (including cytotoxic or helper T celleliciting epitopes), in either eucaryotic or procaryotic recombinantexpression vectors; and the formulation of the same as immunogenicand/or antigenic compositions. Such compositions are described in, forexample, U.S. patent application Ser. No. 08/935,377, the entirecontents of which are incorporated herein by reference. In accordancewith the present invention, the recombinantly expressed C35 epitope maybe expressed, purified and formulated as a subunit vaccine. Preferably,the DNA encoding the C35 epitope may also be constructed into viralvectors, preferably pox virus, adenovirus, herpesvirus, and alphavirusvectors, for use in vaccines. In this regard, either a live recombinantviral vaccine, an inactivated recombinant viral vaccine, or a killedrecombinant viral vaccine can be formulated.

-   -   (i) Expression of C35 in Procaryotic and Eucaryotic Expression        Systems

The present invention encompasses expression systems, both eucaryoticand procaryotic expression vectors, which may be used to express the C35epitope. The C35 epitope may be expressed in both truncated orfull-length forms, in particular for the formation of subunit vaccines.

The present invention encompasses the expression of nucleotide sequencesencoding the C35 polypeptide and immunologically equivalent fragments.Such immunologically equivalent fragments may be identified by makinganalogs of the nucleotide sequence encoding the identified epitopes thatare truncated at the 5′ and/or 3′ ends of the sequence and/or have oneor more internal deletions, expressing the analog nucleotide sequences,and determining whether the resulting fragments immunologically arerecognized by the epitope-specific T lymphocytes and induce acell-mediated immune response, or epitope-specific B lymphocytes forinductions of a humoral immune response.

The invention encompasses the DNA expression vectors that contain any ofthe foregoing coding sequences operatively associated with a regulatoryelement that directs expression of the coding sequences and geneticallyengineered host cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

The C35 epitope gene products or peptide fragments thereof, may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing the C35 epitope gene polypeptidesand peptides of the invention by expressing nucleic acid containingepitope gene sequences are described herein. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing epitope gene product coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., (1989), Cold Spring Harbor Laboratory Press, andAusubel et al., 1989, supra. Alternatively, RNA capable of encodingglycoprotein epitope gene product sequences may be chemicallysynthesized using, for example, synthesizers. See, for example, thetechniques described in “Oligonucleotide Synthesis”, 1984, Gait, M. J.ed., IRL Press, Oxford, which is incorporated by reference herein in itsentirety.

The invention also encompasses nucleotide sequences that encode peptidefragments of the C35 epitope gene products. For example, polypeptides orpeptides corresponding to the extracellular domain of the C35 epitopemay be useful as “soluble” protein which would facilitate secretion,particularly useful in the production of subunit vaccines. The C35epitope gene product or peptide fragments thereof, can be linked to aheterologous epitope that is recognized by a commercially availableantibody is also included in the invention. A durable fusion protein mayalso be engineered; i.e., a fusion protein which has a cleavage sitelocated between the C35 epitope sequence and the heterologous proteinsequence, so that the selected C35 can be cleaved away from theheterologous moiety. For example, a collagenase cleavage recognitionconsensus sequence may be engineered between the C35 epitope protein orpeptide and the heterologous peptide or protein. The epitopic domain canbe released from this fusion protein by treatment with collagenase. In apreferred embodiment of the invention, a fusion protein ofglutathione-S-transferase and the C35 epitope protein may be engineered.

The C35 epitope proteins of the present invention for use in vaccinepreparations, in particular subunit vaccine preparations, aresubstantially pure or homogeneous. The protein is consideredsubstantially pure or homogeneous when at least 60 to 75% of the sampleexhibits a single polypeptide sequence. A substantially pure proteinwill preferably comprise 60 to 90% of a protein sample, more preferablyabout 95% and most preferably 99%. Methods which are well known to thoseskilled in the art can be used to determine protein purity orhomogeneity, such as polyacrylamide gel electrophoresis of a sample,followed by visualizing a single polypeptide band on a staining gel.Higher resolution may be determined using HPLC or other similar methodswell known in the art.

The present invention encompasses C35 polypeptides which are typicallypurified from host cells expressing recombinant nucleotide sequencesencoding these proteins. Such protein purification can be accomplishedby a variety of methods well known in the art. In a preferredembodiment, the C35 epitope protein of the present invention isexpressed as a fusion protein with glutathione-S-transferase. Theresulting recombinant fusion proteins purified by affinitychromatography and the epitope protein domain is cleaved away from theheterologous moiety resulting in a substantially pure protein sample.Other methods known to those skilled in the art may be used; see forexample, the techniques described in “Methods In Enzymology”, 1990,Academic Press, Inc., San Diego, “Protein Purification: Principles andPractice”, 1982, Springer-Verlag, New York, which are incorporated byreference herein in their entirety.

(ii) Eucaryotic and Procaryotic Expression Vectors

The present invention encompasses expression systems, both eucaryoticand procaryotic expression vectors, which may be used to express the C35epitope. A variety of host-expression vector systems may be utilized toexpress the C35 epitope gene of the invention. Such host-expressionsystems represent vehicles by which the C35 coding sequence may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the C35 nucleotide codingsequences, exhibit the C35 epitope gene product of the invention insitu. These include but are not limited to microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the C35 epitope gene product coding sequence; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing the C35 epitope gene product coding sequence; insectcell systems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the C35 epitope gene product coding sequence;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing C35 epitope gene product coding sequence; ormammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

(iii) Host Cells

The present invention encompasses the expression of the C35 epitope inanimal and insect cell lines. In a preferred embodiment of the presentinvention, the C35 epitope is expressed in a baculovirus vector in aninsect cell line to produce an unglycosylated antigen. In anotherpreferred embodiment of the invention, the C35 epitope is expressed in astably transfected mammalian host cell, e.g., CHO cell line to produce aglycosylated antigen. The C35 epitopes which are expressed recombinantlyby these cell lines may be formulated as subunit vaccines. The presentinvention is further directed to host cells that overexpress the C35gene product. The cell may be a host cell transiently or stabletransected or transformed with any suitable vector which includes apolynucleotide sequence encoding the C35 polypeptide or a fragmentthereof and suitable promoter and enhancer sequences to directoverexpression of the C35 gene product. However, the overexpressing cellmay also be a product of an insertion, for example via homologousrecombination, of a heterologous promoter or enhancer which will directoverexpression of the endogenous C35 gene. The term “overexpression”refers to a level of expression which is higher than a basal level ofexpression typically characterizing a given cell under otherwiseidentical conditions.

A host cell strain may be chosen which modulates the expression of theinserted sequences, or modifies and processes the C35 gene product inthe specific fashion desired. Such modifications (e.g., glycosylation)and processing (e.g. cleavage) of protein products may be important forthe function of the protein. Different host cells have characteristicand specific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification of theforeign protein expressed. To this end, eucaryotic host cells whichpossess the cellular machinery for proper processing of the primarytranscript, glycosylation, phosphorylation, and prenylation of the C35gene product may be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 and WI38 celllines.

For long term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe C35 target epitope may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines. This method mayadvantageously be used to engineer cell lines which express the C35epitope gene products. Such cell lines would be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the C35 epitope gene product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

(iv) Expression of C35 Epitope in Recombinant Viral Vaccines

In another embodiment of the present invention, either a liverecombinant viral vaccine or an inactivated recombinant viral vaccineexpressing the C35 epitope can be engineered. A live vaccine may bepreferred because multiplication in the host leads to a prolongedstimulus of similar kind and magnitude to that occurring in naturalinfections, and therefore, confers substantial, long-lasting immunity.Production of such live recombinant virus vaccine formulations may beaccomplished using conventional methods involving propagation of thevirus in cell culture or in the allantois of the chick embryo followedby purification.

In this regard, a variety of viruses may be genetically engineered toexpress the C35 epitope. For vaccine purposes, it may be required thatthe recombinant viruses display attenuation characteristics. Currentlive virus vaccine candidates for use in humans are either cold adapted,temperature sensitive, or attenuated. The introduction of appropriatemutations (e.g., deletions) into the templates used for transfection mayprovide the novel viruses with attenuation characteristics. For example,specific multiple missense mutations that are associated withtemperature sensitivity or cold adaptation can be made into deletionmutations and/or multiple mutations can be introduced into individualviral genes. These mutants should be more stable than the cold ortemperature sensitive mutants containing single point mutations andreversion frequencies should be extremely low. Alternatively,recombinant viruses with “suicide” characteristics may be constructed.Such viruses go through only one or a few rounds of replication in thehost.

For purposes of the invention, any virus may be used in accordance withthe present invention which: (a) displays an attenuated phenotype or maybe engineered to display attenuated characteristics; (b) displays atropism for mammals, in particular humans, or may be engineered todisplay such a tropism; and (c) may be engineered to express the C35epitope of the present invention.

Vaccinia viral vectors may be used in accordance with the presentinvention, as large fragments of DNA are easily cloned into its genomeand recombinant attenuated vaccinia variants have been described (Meyer,et al., 1991, J. Gen. Virol. 72:1031-1038). Orthomyxoviruses, includinginfluenza; Paramyxoviruses, including respiratory syncytial virus andSendai virus; and Rhabdoviruses may be engineered to express mutationswhich result in attenuated phenotypes (see U.S. Pat. No. 5,578,473,issued Nov. 26, 1996). These viral genomes may also be engineered toexpress foreign nucleotide sequences, such as the C35 epitopes of thepresent invention (see U.S. Pat. No. 5,166,057, issued Nov. 24, 1992,incorporated herein by reference in its entirety). Reverse genetictechniques can be applied to manipulate negative and positive strand RNAviral genomes to introduce mutations which result in attenuatedphenotypes, as demonstrated in influenza virus, Herpes Simplex virus,cytomegalovirus and Epstein-Barr virus, Sindbis virus and poliovirus(see Palese et al., 1996, Proc. Natl. Acad. Sci. USA 93:11354-11358).These techniques may also be utilized to introduce foreign DNA, i.e.,the C35 epitopes, to create recombinant viral vectors to be used asvaccines in accordance with the present invention. See, for instance,U.S. patent application Ser. No. 08/935,377, the entire contents ofwhich are incorporated herein by reference. In addition, attenuatedadenoviruses and retroviruses may be engineered to express the C35epitope. Therefore, a wide variety of viruses may be engineered todesign the vaccines of the present invention, however, by way ofexample, and not by limitation, recombinant attenuated vaccinia vectorsexpressing the C35 epitope for use as vaccines are described herein.

In one embodiment, a recombinant modified vaccinia variant, ModifiedVirus Ankara (MVA) is used in a vaccine formulation. This modified virushas been passaged for 500 cycles in avian cells and is unable to undergoa full infectious cycle in mammalian cells (Meyer, et al., 1991, J. Gen.Virol. 72:1031-1038). When used as a vaccine, the recombinant virus goesthrough a single replication cycle and induces a sufficient level ofimmune response but does not go further in the human host and causedisease. Recombinant viruses lacking one or more of essential vacciniavirus genes are not able to undergo successive rounds of replication.Such defective viruses can be produced by co-transfecting vacciniavectors lacking a specific gene(s) required for viral replication intocell lines which permanently express this gene(s). Viruses lacking anessential gene(s) will be replicated in these cell lines but whenadministered to the human host will not be able to complete a round ofreplication. Such preparations may transcribe and translate—in thisabortive cycle—a sufficient number of genes to induce an immuneresponse.

Alternatively, larger quantities of the strains can be administered, sothat these preparations serve as inactivated (killed) virus, vaccines.For inactivated vaccines, it is preferred that the heterologous C35 geneproduct be expressed as a viral component, so that the C35 gene productis associated with the virion. The advantage of such preparations isthat they contain native proteins and do not undergo inactivation bytreatment with formalin or other agents used in the manufacturing ofkilled virus vaccines.

In another embodiment of the invention, inactivated vaccine formulationsare prepared using conventional techniques to “kill” the recombinantviruses. Inactivated vaccines are “dead” in the sense that theirinfectivity has been destroyed. Ideally, the infectivity of the virus isdestroyed without affecting immunogenicity. In order to prepareinactivated vaccines, the recombinant virus may be grown in cell cultureor in the allantois of the chick embryo, purified by zonalultracentrifugation, inactivated by formaldehyde or β-propiolactone, andpooled. The resulting vaccine is usually inoculated intramuscularly.

Inactivated viruses may be formulated with a suitable adjuvant in orderto enhance the immunological response. Such adjuvants may include butare not limited to mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols, polyanions;peptides; oligonucleotides, oil emulsions; and potentially useful humanadjuvants such as BCG and Corynebacterium parvum.

(v) Methods of Treatment and/or Vaccination

Since the C35 epitopes of the present invention can be produced in largeamounts, the antigen thus produced and purified has use in vaccinepreparations. The C35 epitope may be formulated into a subunit vaccinepreparation, or may be engineered into viral vectors and formulated intovaccine preparations. Alternatively, the DNA encoding the C35 epitopemay be administered directly as a vaccine formulation. The “naked”plasmid DNA once administered to a subject invades cells, is expressed,processed into peptide fragments, some of which can be presented inassociation with MHC molecules on the surface of the invaded cell, andelicits a cellular immune response so that T lymphocytes will attackcells displaying the C35 epitope. The C35 epitope also has utility indiagnostics, e.g., to detect or measure in a sample of body fluid from asubject the presence of tumors that express C35 or the presence ofantibodies or T cells that have been induced by C35 expressing tumor andthus to diagnose cancer and tumors and/or to monitor the cellular immuneresponse of the subject subsequent to vaccination.

The recombinant viruses of the invention can be used to treattumor-bearing mammals, including humans, to generate an immune responseagainst the tumor cells. The generation of an adequate and appropriateimmune response leads to tumor regression in vivo. Such “vaccines” canbe used either alone or in combination with other therapeutic regimens,including but not limited to chemotherapy, radiation therapy, surgery,bone marrow transplantation, etc. for the treatment of tumors. Forexample, surgical or radiation techniques could be used to debulk thetumor mass, after which, the vaccine formulations of the invention canbe administered to ensure the regression and prevent the progression ofremaining tumor masses or micrometastases in the body. Alternatively,administration of the “vaccine” can precede such surgical, radiation orchemotherapeutic treatment.

Alternatively, the recombinant viruses of the invention can be used toimmunize or “vaccinate” tumor-free subjects to prevent tumor formation.With the advent of genetic testing, it is now possible to predict asubject's predisposition for certain cancers. Such subjects, therefore,may be immunized using a recombinant vaccinia virus expressing the C35antigen.

The immunopotency of the C35 epitope vaccine formulations can bedetermined by monitoring the immune response in test animals followingimmunization or by use of any immunoassay known in the art. Generationof a cell-mediated and/or humoral immune response may be taken as anindication of an immune response. Test animals may include mice,hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., andeventually human subjects.

Suitable preparations of such vaccines include injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,suspension in, liquid prior to injection, may also be prepared. Thepreparation may also be emulsified, or the polypeptides encapsulated inliposomes. The active immunogenic ingredients are often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine preparation may also include minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and/or adjuvants which enhance the effectiveness ofthe vaccine.

Examples of adjuvants which may be effective, include, but are notlimited to: aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine,GM-CSF, QS-21™ (investigational drug, Progenics Pharmaceuticals, Inc.),DETOX™ (investigational drug, Ribi Pharmaceuticals), BCG, and CpG richoligonucleotides.

The effectiveness of an adjuvant may be determined by measuring theinduction of the cellular immune response directed against the C35epitope.

The vaccines of the invention may be multivalent or univalent.Multivalent vaccines are made from recombinant viruses that direct theexpression of more than one antigen. Multivalent vaccines comprised ofmultiple T cell epitopes, both cytotoxic and helper, are preferred.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc.

Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent. Where thecomposition is administered by injection, an ampoule of sterile diluentcan be provided so that the ingredients may be mixed prior toadministration.

In a specific embodiment, a lyophilized C35 epitope of the invention isprovided in a first container; a second container comprises diluentconsisting of an aqueous solution of 50% glycerin, 0.25% phenol, and anantiseptic (e.g., 0.005% brilliant green).

Use of purified C35 antigens as vaccine preparations can be carried outby standard methods. For example, the purified C35 epitopes should beadjusted to an appropriate concentration, formulated with any suitablevaccine adjuvant and packaged for use. Suitable adjuvants may include,but are not limited to: mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols; polyanions;peptides; oil emulsions; alum, and MDP. The immunogen may also beincorporated into liposomes, or conjugated to polysaccharides and/orother polymers for use in a vaccine formulation. In instances where therecombinant antigen is a hapten, i.e., a molecule that is antigenic inthat it can react selectively with cognate antibodies, but notimmunogenic in that it cannot elicit an immune response, the hapten maybe covalently bound to a carrier or immunogenic molecule; for instance,a large protein such as serum albumin will confer immunogenicity to thehapten coupled to it. The hapten-carrier may be formulated for use as avaccine.

Many methods may be used to introduce the vaccine formulations describedabove into a patient. These include, but are not limited to, oral,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, transdermal, epidural, pulmonary, gastric, intestinal,rectal, vaginal, or urethral routes. When the method of treatment uses alive recombinant vaccinia vaccine formulation of the invention, it maybe preferable to introduce the formulation via the natural route ofinfection of the vaccinia virus, i.e., through a mucosal membrane orsurface, such as an oral, nasal, gastric, intestinal, rectal, vaginal orurethral route, or through the skin. To induce a CTL response, themucosal route of administration may be through an oral or nasalmembrane. Alternatively, an intramuscular or intraperitoneal route ofadministration may be used. Preferably, a dose of 10⁶-10⁷ PFU (plaqueforming units) of cold adapted recombinant vaccinia virus is given to ahuman patient.

The precise dose of vaccine preparation to be employed in theformulation will also depend on the route of administration, and thenature of the patient, and should be decided according to the judgmentof the practitioner and each patient's circumstances according tostandard clinical techniques. An effective immunizing amount is thatamount sufficient to produce an immune response to the antigen in thehost to which the vaccine preparation is administered.

Where subsequent or booster doses are required, a modified vacciniavirus such as MVA can be selected as the parental virus used to generatethe recombinant. Alternatively, another virus, e.g., adenovirus, canarypox virus, or a subunit preparation can be used to boost. Immunizationand/or cancer immunotherapy may be accomplished using a combinedimmunization regimen, e.g., immunization with a recombinant vacciniaviral vaccine of the invention and a boost of a recombinant adenoviralvaccine. In such an embodiment, a strong secondary CD8⁺ T cell responseis induced after priming and boosting with different viruses expressingthe same epitope (for such methods of immunization and boosting, see,e.g., Murata et al., Cellular Immunol. 173:96-107). For example, apatient is first primed with a vaccine formulation of the inventioncomprising a recombinant vaccinia virus expressing an epitope, e.g., aselected tumor-associated antigen or fragment thereof. The patient isthen boosted, e.g., 21 days later, with a vaccine formulation comprisinga recombinant virus other than vaccinia expressing the same epitope.Such priming followed by boosting induces a strong secondary T cellresponse. Such a priming and boosting immunization regimen is preferablyused to treat a patient with a tumor, metastasis or neoplastic growthexpressing the tumor associate, e.g., C35, antigen

In yet another embodiment, the recombinant vaccinia viruses can be usedas a booster immunization subsequent to a primary immunization withinactivated tumor cells, a subunit vaccine containing the C35 antigen orits epitope, or another recombinant viral vaccine, e.g., adenovirus,canary pox virus, or MVA.

In an alternate embodiment, recombinant vaccinia virus encoding C35epitopes or fragment thereof may be used in adoptive immunotherapeuticmethods for the activation of T lymphocytes that are histocompatiblewith the patient and specific for the C35 antigen (for methods ofadoptive immunotherapy, see, e.g., Rosenberg, U.S. Pat. No. 4,690,915,issued Sep. 1, 1987; Zarling, et al., U.S. Pat. No. 5,081,029, issuedJan. 14, 1992). Such T lymphocytes may be isolated from the patient or ahistocompatible donor. The T lymphocytes are activated in vitro byexposure to the recombinant vaccinia virus of the invention. Activated Tlymphocytes are expanded and inoculated into the patient in order totransfer T cell immunity directed against the C35 antigen epitope.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers comprising one or more of the ingredients of thevaccine formulations of the invention. Associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

Cancer Diagnosis and Prognosis

There are two classes of genes affecting tumor development. Genesinfluencing the cancer phenotype that act directly as a result ofchanges (e.g., mutation) at the DNA level, such as BRCA1, BRCA2, andp53, are one class of genes. Another class of genes affect the phenotypeby modulation at the expression level. Development of breast cancer andsubsequent malignant progression is associated with alterations of avariety of genes of both classes. Identification of quantitative changesin gene expression that occur in the malignant mammary gland, ifsufficiently characterized, may yield novel molecular markers which maybe useful in the diagnosis and treatment of human breast cancer.

The present inventors have identified a new breast cancer marker, C35,that is differentially expressed in primary infiltrating intraductalmammary carcinoma cells. Low expression levels of C35 in normal mammaryepithelial cells suggest that overexpression of C35 indicates breastcancer malignant progression. It is possible that C35 may also beoverexpressed in tumors of certain other tissue types including bladderand lung.

The present inventors have demonstrated that certain tissues in mammalswith cancer express significantly enhanced levels of the C35 protein andmRNA encoding the C35 protein when compared to a corresponding“standard” mammal, i.e., a mammal of the same species not having thecancer. Further, it is believed that enhanced levels of the C35 protein,or of antibodies or lymphocytes specific for the C35 protein, can bedetected in certain body fluids (e.g., sera, plasma, urine, and spinalfluid) from mammals with cancer when compared to sera from mammals ofthe same species not having the cancer. Thus, the present inventionprovides a diagnostic method useful for tumor diagnosis, which involvesassaying the expression level of the gene encoding the C35 protein inmammalian cells or body fluid and comparing the gene expression levelwith a standard C35 gene expression level, whereby an increase in thegene expression level over the standard is indicative of certain tumors.Alternatively, the expression levels of antibodies or lymphocytesspecific for C35 protein or C35 polypeptides can be determined in bloodor other body fluids and compared with a standard of expression ofC35-specific antibodies or lymphocytes.

Where a tumor diagnosis has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting enhanced C35 gene expression may experiencea worse clinical outcome relative to patients expressing the gene at alower level.

By “assaying the expression level of the gene encoding the C35 protein”is intended qualitatively or quantitatively measuring or estimating thelevel of the C35 protein or the level of the mRNA encoding the C35protein in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level or mRNA level) orrelatively (e.g., by comparing to the C35 protein level or mRNA level ina second biological sample).

Preferably, the C35 protein level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard C35 proteinlevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the cancer. As will beappreciated in the art, once a standard C35 protein level or mRNA levelis known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source which containsC35 protein or mRNA. Biological samples include mammalian body fluids(such as sera, plasma, urine, synovial fluid and spinal fluid) whichcontain secreted mature C35 protein, and ovarian, prostate, heart,placenta, pancreas, liver, spleen, lung, breast, bladder and umbilicaltissue which may contain precursor or mature forms of C35.

The present invention is useful for detecting cancer in mammals. Inparticular, the invention is useful during diagnosis of the followingtypes of cancers in mammals: breast, bladder, ovarian, prostate, bone,liver, lung, pancreatic, and splenic. Preferred mammals include monkeys,apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularlypreferred are humans.

Total cellular RNA can be isolated from a biological sample using thesingle-step guanidinium-thiocyanate-phenol-chloroform method describedin Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the C35 protein are then assayed using any appropriatemethod. These include Northern blot analysis (Harada et al., Cell63:303-312 (1990)), S1 nuclease mapping (Fujita et al., Cell 49:357-367(1987)), the polymerase chain reaction (PCR), reverse transcription incombination with the polymerase chain reaction (RT-PCR) (Makino et al.,Technique 2:295-301 (1990)), and reverse transcription in combinationwith the ligase chain reaction (RT-LCR).

Assaying C35 protein levels in biological sample can occur usingantibody-based techniques. For example, C35 protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)).

Other antibody-based methods useful for detecting C35 protein expressioninclude immunoassays, such as enzyme linked immunosorbent assay (ELISA),ELISPOT, and the radioimmunoassay (RIA).

Suitable labels are known in the art and include enzyme labels, such as,Glucose oxidase, and radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium(^(99m)Tc), and fluorescent labels, such as fluorescein and rhodamine,and biotin.

C35-specific T cells may be detected in a variety of proliferation andlymphokine secretion assays following activation by C35 presented byantigen presenting cells according to methods known in the art.Tetrameric complexes of a C35 peptide epitope bound to soluble MHCmolecules can be employed to directly stain and enumerate C35-specific Tcells in a population of cells (Lee, P. P. et al., Nature Medicine5:677-85 (1999) the entire contents of which is hereby incorporated byreference.

In addition to assaying secreted protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byXradiography, NMR or ESR. For Xradiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A proteinspecific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, ¹³¹I, ¹¹²In, ⁹⁹mTc), a radioopaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99 mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Fusion Proteins

Any C35 polypeptide can be used to generate fusion proteins. Forexample, the C35 polypeptide, when fused to a second protein, can beused as an antigenic tag. Antibodies raised against the C35 polypeptidecan be used to indirectly detect the second protein by binding to theC35. Moreover, because secreted proteins target cellular locations basedon trafficking signals, the C35 polypeptides can be used as a targetingmolecule once fused to other proteins. As used herein, the term “fusionprotein” does not mean C35 polypeptide fragments.

Examples of domains that can be fused to C35 polypeptides include notonly heterologous signal sequences, but also other heterologousfunctional regions. The fusion does not necessarily need to be direct,but may occur through linker sequences. In a preferred embodiment, thefusion protein of the present invention comprises at least one C35peptide epitope or C35 peptide epitope analog joined to at least oneadditional C35 peptide epitope or C35 peptide epitope analog. In aparticularly preferred embodiment, the fusion proteins of the presentinvention comprise homopolymers of the same C35 peptide epitope or C35peptide epitope analog, as well as heteropolymers of different C35peptide epitopes and C35 peptide epitope analogs.

In certain preferred embodiments, C35 fusion polypeptides may beconstructed which include additional Nterminal and/or Cterminal aminoacid residues. In particular, any Nterminally or Cterminally deleted C35polypeptide disclosed herein may be altered by inclusion of additionalamino acid residues at the Nterminus to produce a C35 fusionpolypeptide. In addition, C35 fusion polypeptides are contemplated whichinclude additional Nterminal and/or Cterminal amino acid residues fusedto a C35 polypeptide comprising any combination of N- and Cterminaldeletions set forth above.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the C35 polypeptide. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the Nterminus of the C35 polypeptide to improve stability andpersistence during purification from the host cell or subsequenthandling and storage. Also, peptide moieties may be added to the C35polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the C35 polypeptide. The addition ofpeptide moieties to facilitate handling of polypeptides are familiar androutine techniques in the art.

Moreover, C35 polypeptides, including fragments, and specificallyepitopes, can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased halflife in vivo.One reported example describes chimeric proteins consisting of the firsttwo domains of the human CD4polypeptide and various domains of theconstant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:8486(1988).) Fusion proteins having disulfidelinked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:39583964 (1995).)

Similarly, EPAO 464 533 (Canadian counterpart 2045869) discloses fusionproteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EPA 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL5, have been fused with Fc portions for the purpose of highthroughputscreening assays to identify antagonists of hIL5. (See, D. Bennett etal., J. Molecular Recognition 8:5258 (1995); K. Johanson et al., J.Biol. Chem. 270:94599471 (1995).)

Moreover, the C35 polypeptides can be fused to marker sequences, such asa peptide which facilitates purification of C35. In preferredembodiments, the marker amino acid sequence is a hexahistidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821824 (1989), for instance, hexahistidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984).)

Thus, any of these above fusions can be engineered using the C35polynucleotides or the C35 polypeptides.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the C35polynucleotide, host cells, and the production of C35 polypeptides byrecombinant techniques. The vector may be, for example, a phage,plasmid, viral, or retroviral vector. Retroviral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.

C35 polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The C35 polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabovedescribed host cells are known in the art.

Among vectors preferred for use in bacteria include pHE-4 (and variantsthereof); pQE70, pQE60 and pQE9, available from QIAGEN, Inc.;pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK2233,pKK2333, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Amongpreferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; and pSVK3, pBPV, pMSG and pSVL available fromPharmacia. Other suitable vectors will be readily apparent to theskilled artisan. Preferred vectors are poxvirus vectors, particularlyvaccinia virus vectors such as those described in U.S. patentapplication Ser. No. 08/935,377, the entire contents of which areincorporated herein by reference.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAEdextran mediated transfection,cationic lipidmediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

C35 polypeptides can be recovered and purified from recombinant cellcultures by wellknown methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

C35 polypeptides can also be recovered from: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect, and mammalian cells. Depending upon thehost employed in a recombinant production procedure, the C35polypeptides may be glycosylated or may be nonglycosylated. In addition,C35 polypeptides may also include an initial modified methionineresidue, in some cases as a result of hostmediated processes. Thus, itis well known in the art that the Nterminal methionine encoded by thetranslation initiation codon generally is removed with high efficiencyfrom any protein after translation in all eukaryotic cells. While theNterminal methionine on most proteins also is efficiently removed inmost prokaryotes, for some proteins, this prokaryotic removal process isinefficient, depending on the nature of the amino acid to which theNterminal methionine is covalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g. C35 coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with C35 polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous C35 polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous C35 polynucleotide sequences via homologous recombination(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication No. WO 96/29411, published Sep. 26, 1996; InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al.,Nature 342:435-438 (1989), the disclosures of each of which areincorporated by reference in their entireties).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Differential Expression of C35 in Human BreastCarcinoma

The present inventors have characterized a full-length cDNA representinga gene, C35, that is differentially expressed in human breast andbladder cancer (FIG. 1A). A 348 base pair DNA fragment of C35 wasinitially isolated by subtractive hybridization of poly-A RNA from tumorand normal mammary epithelial cell lines derived from the same patientwith primary infiltrating intraductal mammary carcinoma. (Band, V. etal., Cancer Res. 50:7351-7357 (1990). Employing primers based on thissequence and that of an overlapping EST sequence (Accession No. W57569),a cDNA that includes the full-length C35 coding sequence was thenamplified and cloned from the SKBR3 breast tumor cell line (ATCC,HTB-19). This C35 cDNA includes, in addition to the 348 bp codingsequence, 167 bp of 3′ untranslated region.

Differential expression of the C35 sequence is demonstrated in FIG. 2Awhich compares expression levels of clone C35 in poly-A RNA from celllines derived from normal mammary epithelium, from two primary breasttumor nodules, and from two metastatic lung tumor nodules isolatedapproximately one year later from the same patient (Band, V. et al.,Cancer Res. 50:7351-7357 (1990)). Quantitative analysis indicates thatthe sequence is expressed at a more than 10 fold higher level in tumorcells than in normal mammary epithelium. Low expression levels in apanel of other normal tissues is demonstrated by the Northernhybridization results of FIG. 2B. Even though three times as much poly-ARNA was loaded from normal tissues as from the tumor cell lines, littleor no expression of RNA homologous to C35 was detected after acomparable 15 hour exposure. Only after an extended 96 hour exposure waslow level expression of some homologous sequences detected in normalspleen and kidney tissues. Analysis of expression of C35 homologoussequences in poly-A RNA from three primary infiltrating ductal breastcarcinoma from different patients as well as a sample of normal breastepithelium is shown in FIG. 2C. In comparison to normal breastepithelium, sequences homologous to C35 are overexpressed as much as 45and 25 fold in two of the three primary breast tumors.

The present inventors previously conducted an analysis of animmunoprotective tumor antigen expressed in several independentlyderived murine tumors and, at much reduced levels, in normal mousetissues. (See U.S. patent application filed Mar. 28, 2000, titled“Methods of Producing a Library and Methods of Directly Selecting CellsExpressing Inserts of Interest,” the entire contents of which are herebyincorporated herein by reference). In this case, a factor of 9difference between expression levels in tumor and normal tissues wasassociated with induction of an immunoprotective tumor-specificresponse. As discussed above, the expression level of C35 in some humanbreast cancers relative to normal tissue exceeds a factor of 9,suggesting that C35 might also be immunoprotective against breast cancerin these individuals.

Example 2 C35 Specific CTL are Cytolytic for C35 Positive Breast TumorCells

Although a gene product may be overexpressed in tumor cells, as is thecase for C35, it is immunologically relevant only if peptides derivedfrom that gene product can be processed and presented in associationwith MHC molecules of the tumor cells. It is conceivable that for anygiven gene product either no peptides are produced during the cellulardegradation process that satisfy the requirements for binding to the MHCmolecules expressed by that tumor, or, even if such peptides aregenerated, that defects in transport or competition for MHC molecules byother tumor peptides would preclude presentation of any peptides fromthat specific gene product. Even if relevant tumor peptides areprocessed and presented in association with human MHC in the tumorcells, it must in all cases be determined whether human T cells reactiveto these peptides are well represented in the repertoire or whether Tcells may have been rendered tolerant, perhaps due to expression of thesame or a related antigen in some other nonhomologous normal tissue. Forboth these reasons, therefore, it is essential to confirm thatMHCrestricted, human tumor antigenspecific T cells can be induced by C35and that they are indeed crossreactive on human tumor cells. Relevantinformation on this point can be obtained through in vitro stimulationof human T cell responses with recombinant C35 or C35 peptides presentedby autologous antigen presenting cells.

A major technical problem in evaluating T cell responses to recombinantgene products is that a strong immune response against the expressionvector can block or obscure the recombinant specific response. This isparticularly a problem with primary responses that may require multiplecycles of in vitro stimulation. To minimize vector specific responses,it is possible to alternate stimulation by antigen presenting cellsinfected with different viral vectors recombinant for the same geneproduct. Convenient vectors include: retroviruses, adenovirus, and poxviruses.

Human PBMC were purified using Ficoll-Hypaque and subjected to rosettingwith neuraminidasetreated sheep erythrocytes to isolate monocytes(erythrocyte rosette negative, ER) and T lymphocytes (ER⁺). Dendriticcells were generated from the ER fraction by culture for 7 days inrhGMCSF (1000 U/ml) and rhIL4 (1000 U/ml) with fresh medium andcytokines being added every other day. At day 7, immature dendriticcells were transduced with retrovirus expressing human C35 in thepresence of polybrene (1 ug/ml) for 6 hours. Cells were washed andincubated under maturation conditions for 4 days in the presence of12.5% monocyte conditioned medium, 1000 U/ml rhGMCSF and 1000 rhU/ml IL4and 1% autologous serum. At this point, the dendritic cells wereincubated with autologous T lymphocytes (cryopreserved ER⁺ fraction) ata ratio of 1 DC:50 T cells for 14 days. Viable T cells were restimulatedwith autologous, irradiated EBVB B cells infected at a multiplicity ofinfection of 1 overnight (16 hours) with a vaccinia recombinantexpressing human C35 in the presence of cytokines IL2 (20 U/ml), IL12(20 U/ml) and IL18 (10 ng/ml). Cells were restimulated two more timeswith autologous EBVB cells infected with C35bearing retrovirus in thepresence of IL2 and IL7 (10 ng/ml). Cytotoxic activity was measuredafter a total of 4 stimulations by ⁵¹Cr release assay using 5000targets/well in a 4 hour assay. The results shown in Table 8 belowdemonstrate specific cytotoxic activity of C35 stimulated T cellsagainst 21NT breast tumor cells that express relatively elevated levelsof C35 but not against MDAMB231 tumor cells that express the same lowlevels of C35 as normal nontransformed epithelial cells.

TABLE 8 C35 specific CTL are Cytolytic for C35 Positive Breast TumorCells Target E:T Cells HLA Haploype 20:1 10:1 Autologous (Effectors: A2,A11; B8, B35) (% specific lysis) EBVB A2, A11; B8, B35 2 1 MDAMB231 A2;B8 3 1 C35 low (1x) 21NT A26, A31; B35, B38 22 10 C35 high (12x) K562 20

Example 3 C35 Expression on the Membrane of Breast Carcinoma Cells

To determine whether the C35 polypeptide product is expressed on thesurface of tumor cells, a C35 specific antiserum was prepared. BALB/cmice were immunized with syngeneic Line 1 mouse tumor cells that hadbeen transduced with retrovirus encoding human C35. Mice were bledfollowing a series of two or more immunizations. The immune sera wereemployed to detect surface expression of C35 protein by flow cytometryon three breast tumor cell lines representing high (21NT), intermediate(SKBR3), and low (MDA-MB-231 levels of expression of the C35 transcriptin Northern blots (see FIGS. 4A-4C). 1×10⁵ breast tumor cells werestained with 3.5 microliters of C35 specific antiserum or control,pre-bleed BALB/c serum. After a 30 minute incubation, cells were washedtwice with staining buffer (PAB) and incubated with FITC-goat anti-mouseIgG (1 ug/sample) for 30 minutes. Samples were washed and analyzed on anEPICS ELITE™ flow cytometer. The results presented in FIGS. 4A-4Cdemonstrate membrane expression of the C35 antigen recognized by thespecific immune serum at high levels on tumor line 21NT (FIG. 4A),intermediate levels for tumor line SKBR3 (FIG. 4B), and undetectablelevels in tumor line MDA-MB-231 (FIG. 4C). The high level of reactivityof antibody to membranes of tumor cells that express elevated levels ofC35 transcripts suggests that C35 specific antibodies may serve aseffective immunotherapeutic agents for the large number of breastcarcinoma that overexpress this gene product (see FIGS. 2A-2C and 3).

Example 4 A Deregulated Ribosomal Protein L3 Gene Encodes a SharedMurine Tumor Rejection Antigen

The present inventors have developed novel antigen discovery technologythat allows for the selection of genes encoding CTL epitopes from a cDNAlibrary constructed in a poxvirus. Using this technology the presentinventors have determined that a shared murine tumor antigen is encodedby an alternate allele of the ribosomal protein L3 gene. The immunogenicL3 gene is expressed at significant albeit reduced levels in normaltissues including thymus. Immunization with a vaccinia recombinant ofthe immunogenic L3 cDNA induces protective immunity against tumorchallenge. It is of particular interest that a deregulated allele of ahousekeeping gene can serve as an immunoprotective antigen and thatthymic expression does not preclude immunogenicity of an upregulatedtumor product. These observations emphasize that tolerance to aself-protein is not absolute but must be defined in relation toquantitative levels of expression. The ribosomal protein described maybe representative of a class of shared tumor antigens that arise as aresult of deregulated expression of a self-protein without compromisingimmune tolerance to normal tissues. Such antigens would be suitable forimmunotherapy of cancer in vital organs.

Methods

Total RNA was isolated from BCA 39 tumor cells using the PERFECT RNA™Total RNA Isolation Kit (5 Prime 3 Prime, Inc., Boulder, Colo.). Poly A+mRNA was isolated from the total RNA using DYNABEADS™ (Dynal, LakeSuccess, N.Y.). Two micrograms of poly A+ mRNA was converted to doublestranded cDNA using the GREAT LENGTHS cDNA SYNTHESIS KIT™ (Clontech,Palo Alto, Calif.). The double stranded cDNA was then inserted invaccinia virus vector v7.5/tk.

Balb/cByJ (Jackson Labs) mice were immunized intraperitoneally with2×10⁶ irradiated (6,500 cGy) BCA 34 cells. Two weeks later the mice wereboosted by subcutaneous injection of 2×10⁶ irradiated BCA 34 cells. Oneweek following the second immunization splenocytes were harvested,divided into 12 parts and cultured in 12 well plates with 6×10⁵irradiated (10,000 cGy), mitomycin C treated BCA 34 cells per well. Atweekly intervals viable T cells were purified using Lympholyte-M(Accurate Chemical, Westbury, N.Y.) and cultured in 12 well plates at1.5×10⁶ T cells per well. To each well was also added 4×10⁶ irradiated(5000 cGy) Balb/c spleen, along with 6×10⁵ irradiated, mitomycin Ctreated BCA 34 cells.

A specific vaccinia recombinant that encodes the well characterizedovalbumin 257-264 peptide (SIINFEKL (SEQ ID NO:2122)) that isimmunodominant in association with H-2K^(b) was diluted withnonrecombinant virus so that it initially constituted either 0.2%,0.01%, or 0.001% of total viral pfu. An adherent monolayer of MC57Gcells (H 2^(b)) were infected with this viral mix at m.o.i.=1(approximately 5×10⁵ cells/well). Following 12 hours infection,ovalbumin peptidespecific CTL, derived by repeated in vitro stimulationof ovalbumin primed splenic T cells with the immunodominant SIINFEKL(SEQ ID NO:2122) peptide, were added. During this incubation thoseadherent cells which were infected with a recombinant particle thatexpresses the ovalbumin peptide are targeted by specific cytotoxic Tcells and undergo a lytic event which causes them to be released fromthe monolayer. Following incubation with CTL, the monolayer is gentlywashed, and both floating cells and the remaining adherent cells areseparately harvested. Virus extracted from each cell population wastitred for the frequency of recombinant (BRdU resistant) viral pfu.Virus extracted from floating cells was then used as input to anotherenrichment cycle with fresh adherent MC57G cells and ovalbuminpeptide-specific CTL. It was observed that following enrichment of VVovato greater than 10% of total virus, further enrichment of therecombinant virus was accelerated if the m.o.i. in succeeding cycles wasreduced from 1 to 0.1.

Confluent monolayers of BCN in wells of a 12 well plate were infectedwith moi=1.0 vaccinia BCA39 cDNA library. At 12 hours post-infection themonolayers were washed 3× with media, and 2.5×10⁶ CTL were added to thewells in a 250 μl volume. The T cells and targets were incubated at 37°C. for 4 hours. Following the incubation the supernatant was harvested,and the monolayer gently washed 3× with 250 μl media. Virus was releasedfrom the cells by freeze/thaw, and titers determined by plaque assay onBSC1 cells. The selected virus population (floating cells in culturesthat received specific T cells) was amplified on BSC1 cells in one wellof a 12 well plate for 2 days. The virus was then harvested and titered.This viral stock was subjected to three additional enrichment cycles.The selected virus population was not amplified prior to the next cycle.

Virus from the fourth enrichment cycle was divided into 40 pools of 5pfu each. Each pool was amplified on BSC1 cells in a 96 well plate, with1 pool/well. After 4 days the virus was harvested (P1), and used toinfect monolayers of BCN in a 96 well plate at moi=5, with 1 pool perwell. As a control, a monolayer of BCN was infected with moi=5 vNotI/tk(Merschlinsky et al., Virology 190:522 (1992)). At 5 hourspost-infection, 2×10⁴ washed CTL were added to each well. The finalvolume in each well was 225 μl. The cells were incubated at 37° C. for18 hours. The cells were then pelleted by centrifugation, 150 μlsupernatant was harvested and tested for IFNg by ELISA. Twenty seven ofthe forty pools of 5 pfu were positive for the ability to stimulate CTL.Suggesting, by Poisson analysis, that specific recombinants wereenriched to greater than 20%. Individual clones were picked from 5positive pools and assayed as above.

Monolayers of B/C.N in a 6 well plate were infected with moi=1.0 ofv7.5/tk, vF5.8, or vH2.16. At 14 hours post-infection cells wereharvested along with the control targets: B/C.N, BCA 34, and BCA 39. Thetarget cells were labeled with 100 microcuries ⁵¹Chromium (Dupont,Boston, Mass.) for 1 hour at 37° C., and 10⁴ cells were added to wellsof a 96 well round bottom plate in quadruplicate. Tumor specific CTLwere added to target cells at the indicated ratios. Cells were incubatedat 37° C. for 4 hours. Supernatants were harvested and ⁵¹Cr releasedetermined. Spontaneous release was derived by incubating target cellswith media alone. Maximal release was determined by incubating targetcells with 5% Triton X 100. Percentage of specific lysis was calculatedusing the formula: % specific lysis=((experimental release−spontaneousrelease)/(maximal release−spontaneous release))×100. In each case themean of quadruplicate wells was used in the above formula.

Two micrograms of total RNA was converted to cDNA using a dT primer andSUPERSCRIPT™ II Reverse Transcriptase (BRL, Gaithersburg, Md.). cDNA wasused as the template for a PCR using L3 specific primers; L3.F1.S(CGGCGAGATGTCTCACAGGA SEQ ID NO:2124)) and L3.F1.AS(ACCCCACCATCTGCACAAAG (SEQ ID NO:2125)); and KLENTAQ™ DNA Polymerase Mix(Clontech) in a 20 microliter final volume. Reaction conditions includedan initial denaturation step of 94° C. for 3 minutes, followed by 30cycles of: 94° C. 30 seconds, 60° C. for 30 seconds, 68° C. for 2minutes. These PCR products contained the region of L3 between position3 and 1252. The PCR products were purified using CENTRICON™ 100 columns(Amicon, Beverly, Mass.), digested with Sau3AI, and resolved on a 3%Agarose/ethidium bromide gel.

Adult female Balb/cByJ mice (2 mice per group) were immunized bysubcutaneous injection of 5×10⁶ pfu of vH2.16, or v7.5/tk. Seven daysfollowing the immunization splenocytes were harvested and cultured in 12well plates along with 1 micromolar peptide L3₄₈₋₅₆(I54). After sevendays the viable T cells were purified using Lympholyte-M, and 1×10⁶ Tcells were added to wells of a 12 well plate along with 1 micromolarpeptide and 4×10⁶ irradiated (5000 cGy) Balb/c spleen cells per well.

Adult female Balb/cByJ mice were immunized by subcutaneous injection of10×10⁶ pfu of vH2.16, vPKIa, v7.5/tk or Phosphate Buffered Saline.Secondary immunizations were given 21 days later. Mice were challengedwith tumor by subcutaneous injection of 2×10⁵ BCA 34 cells twenty one(primary immunization only) or fourteen days following immunization.

Results and Discussion

Prospects for development of broadly effective tumor vaccines have beenadvanced by evidence that several self-proteins can be recognized astumor antigens by immune T cells (Van den Eynde et al., J. Exp. Med.173:1373 (1991); M. B. Bloom et al., J. Exp. Med. 185:453 (1997); VanDer Bruggen et al., Science 254:1643 (1991); Gaugler et al., J. Exp.Med. 179:921 (1994); Boel et al., Immunity 2:167 (1995); Van Den Eyndeet al., J. Exp. Med. 182:689 (1995); Kawakami et al., Proc. Natl. Acad.Sci. U.S.A. 91:3515 (1994); Kawakami et al., Proc. Natl. Acad. Sci.U.S.A. 91:6458 (1994); Brichard et al., J. Exp. Med. 178:489 (1993)).Such normal, nonmutated gene products may serve as common targetantigens in tumors of certain types arising in different individuals.Clinical evidence for induction of protective immunity followingvaccination with such shared tumor antigens is, currently, very limited(Marchand et al., Int. J. Cancer 80:219 (1999); Rosenberg et al., Nat.Med. 4:321 (1998); Overwijk et al., Proc. Natl. Acad. Sci. 96:2982(1999); Brandle et al., Eur. J. Immunol. 28:4010 (1998)). It is,moreover, not at all clear whether the T cell responses to theseself-proteins represent a surprising breakdown in immunologicaltolerance or are a consequence of qualitative or quantitative changes inthe expression of the self-proteins in tumor cells. In the latter case,normal tissue tolerance could be maintained and vaccine induced immunityto self-proteins whose expression is systematically altered in tumorsmight be applicable even to cancer of vital organs.

The present inventors have shown that a ribosomal protein allele that issystematically deregulated in multiple murine tumors during thetransformation process is a tumor rejection antigen and that theprincipal correlate of immunogenicity is a dramatic change inquantitative expression in tumors relative to normal tissues and thymus.

Previously, the present inventors have reported that cross-protectiveimmunity is induced among three independently derived murine tumor celllines (Sahasrabudhe et al., J. Immunology 151:6302 (1993)). Thesetumors, BCA 22, BCA 34, and BCA 39 were derived by in vitro mutagenesisof independent subcultures of the B/C.N line, a cloned, immortalized,anchorage-dependent, contact inhibited, nontumorigenic fibroblast cellline derived from a Balb/c embryo (Collins et al., Nature 299:169(1982); Lin et al., JNCI 74:1025 (1985)). Strikingly, immunization withany of these tumor cell lines, but not with B/C.N provided protectionagainst challenge with not only homologous tumor cells, but also againstchallenge with the heterologous tumor cell lines. Following immunizationwith any of these three tumor cell lines, CD8+ cytolytic T lymphocyte(CTL) lines and clones could be generated which in vitro displayedcrossreactive specificity for the same three tumors, but not for thenon-tumorigenic B/C.N cells from which they derived.

In order to move from an immunological definition to a moleculardefinition of this shared tumor antigen(s), the present inventorsdeveloped a novel and efficient method for the identification of genesthat encode CTL target epitopes. In this approach a cDNA library fromthe BCA 39 tumor cell line was constructed in a modified vaccinia virusexpression vector (Merchlinsky et al., Virology 238:444 (1997); E. Smithet al., Manuscript in preparation). Five hundred thousand plaque formingunits (pfu) of this library were used to infect a monolayer ofantigen-negative B/C.N cells at a multiplicity of infection (moi) of 1.Following 12 hours infection, BCA 34 tumor specific CTL were added tothe target cell monolayer at an effector to target ratio that givesapproximately 50% lysis in a standard ⁵¹Cr release assay. CTL specificfor the heterologous BCA 34 tumor cell line were used in order tofacilitate the identification of antigen(s) which are shared betweenthese two tumor cell lines. Since adherence is an energy dependentprocess, it was expected that cells that undergo a CTL mediated lyticevent would come off of the monolayer and could be recovered in thesupernatant. By harvesting virus from floating cells following cellmediated lymphocytotoxicity (CML), it was possible to enrich for viralrecombinants that had sensitized the host cell to lysis. An essentialfeature of this procedure is that it lends itself to repetition. Thevirus harvested following one cycle of enrichment can be used as inputfor additional cycles of selection using fresh monolayers and fresh CTLuntil the desired level of enrichment has been achieved. In a modelexperiment with CTL specific for a known recombinant, it was possible todemonstrate that specific recombinants could be enriched from an initialdilution of 0.001% to approximately 20% in 6 cycles of selection (Table9). At this level it is a simple matter to pick individual plaques forfurther characterization.

TABLE 9 Multiple Cycles of Enrichment for VVova Enrichment % VVova inFloating Cells Cycle # Expt. 1 Expt. 2 Expt. 3 moi = 1 0 0.2 0.01 0.0011 2.1 0.3 nd 2 4.7 1.1 nd 3 9.1 4.9 nd 4 14.3 17.9 1.4 5 24.6 3.3 6 18.6moi = 0.1 5 48.8 39.3 A vaccinia cocktail composed of wild type vNotl/tk(tk+) spiked with the indicated concentrations of VVova (tk−) wassubjected to CML Selection (12) % VVova = (Titer with BudR/Titer withoutBudR) × 100 nd = not determined

The poxvirus expression library was subjected to 4 cycles of selectionwith tumor-specific CTL. Individual plaques of the selected viralrecombinants were expanded and used to infect separate cultures of B/C.Ncells. These cells were assayed for the ability to stimulate specificCTL to secrete interferon gamma (IFN-gamma) (FIG. 5A), or forsensitization to lysis by the tumor-specific CTL (FIG. 5B). Ten viralclones were isolated, all of which conferred upon B/C.N the ability tostimulate a line of tumor-specific CTL to secrete IFNγ. All 10 clonescontained the same sized (1,300 bp) insert (Smith et al., unpublisheddata). Sequence analysis confirmed that clones F5.8 and H2.16 containedthe same full-length cDNA. It appeared, therefore, that all ten cloneswere recombinant for the same cDNA. In all, 6 of 6 CTL lines that weregenerated by immunization with BCA 34 demonstrated specificity for thisantigen.

A search of GenBank revealed that this cDNA is highly homologous to themurine ribosomal protein L3 gene (Peckham et al., Genes and Development3:2062 (1989)). Sequencing the entire H2.16 clone revealed only a singlenucleotide substitution that coded for an amino acid change whencompared to the published L3 gene sequence. This C170T substitutiongenerates a Threonine to Isoleucine substitution at amino acid position54. The F5.8 clone also contained this nucleotide substitution.

Since CTL recognize antigen as peptide presented by a MajorHistocompatibility Complex (MHC) molecule, it was of interest toidentify the peptide epitope recognized by these class I MHC-restrictedtumor-specific CD8+ T cells. It was considered likely that the alteredamino acid (Ile 54) would be included in the peptide recognized by theCTL. This hypothesis was supported by the demonstration that a vacciniavirus clone recombinant for only the first 199 bp (63 amino acids) ofH2.16 (vH2₁₉₉) was able to sensitize B/C.N to lysis by tumor-specificCTL (Smith et al., unpublished data). A Computer screen ofpeptide-binding motifs suggested that there are two epitopes encodedwithin this region that could associate with high affinity to the classI MHC molecule Kd (FIG. 12) (Parker et al., J. Immunology 152:163(1994)). These two peptides, L3₄₅₋₅₄ (I54) and L3₄₈₋₅₆ (I54) weresynthesized and tested for the ability to sensitize B/C.N cells to lysisby tumor-specific CTL. As shown in FIG. 7A, peptide L3₄₈₋₅₆ (I54)sensitized B/C.N to lysis, while L3₄₅₋₅₄ (I54), and the wild typeL3₄₈₋₅₆ (T54) did not. It was determined that 10 nM L3₄₈₋₅₆ (I54) wassufficient to sensitize targets to lysis by CTL, whereas 100 mM L3₄₈₋₅₆(T54) did not (FIG. 7B). These results demonstrate that peptide L3₄₈₋₅₆(I54) is a target epitope recognized by the tumor-specific CTL.

To analyze expression of the different L3 gene products, oligo-dT primedcDNA was synthesized from RNA of tumors and the B/C.N cell line fromwhich they derived. The first strand cDNA was subjected to PCRamplification using a pair of primers which amplify nearly the entiremouse L3 mRNA. Sequence analysis of these PCR products showed that B/C.Nand BCB13 L3 cDNA contained a C at position 170 (same as publishedsequence). BCB13 is a tumor cell line that was derived from the B/C.Ncell line, but that is not immunologically cross-protective with the BCAtumor cell lines (Sahasrabudhe et al., J. Immunology 151:6302 (1993)).Sequence analysis of the PCR products from the crossreactive BCA 39, BCA34, and BCA 22 tumors suggested that these cell lines express twodifferent species of L3 mRNA. One species contains a C at 170, and theother contains a T at 170, as in the H2.16 clone. The sequence of all L3cDNAs were identical except for this one base substitution.

There are two possible ways to account for the origin of the new L3 RNAin tumor cells. Either the L3 (C170T) gene expressed in these tumors isa somatic mutant of the wild type gene or there are multiple germ linealleles of L3, at least one of which gives rise to an immunogenicproduct when deregulated during the process of tumor transformation. Weconsidered the first hypothesis unlikely because the crossreactive BCA39, BCA 34, and BCA 22 tumors were independently derived. It would beremarkable if the same mutant epitope was generated in all three tumors.On the other hand, Southern blots of different restriction digests ofgenomic DNA from BCA 39 and B/C.N suggested that there are multiplecopies of the L3 gene in the mouse genome (Smith et al., unpublisheddata). The L3 gene has also been reported to be multi-allelic in boththe rat and the cow (Kuwano et al., Biochemical and Biophysical ResearchCommunications 187:58 (1992); Simonic et al., Biochemica et BiophysicaActa 1219:706 (1994)). Further analysis was required to test thehypothesis that different L3 alleles in the germ line are subject todifferential regulation in tumors and normal cells.

The nucleotide sequence of the published L3 from position 168 to 171 isGACC. The sequence of H2.16 in this same region is GATC (FIG. 8A). Thisnew palindrome is the recognition sequence for a number of restrictionendonucleases, including Sau3AI. As shown in the restriction map of FIG.8A, a Sau3A I digest of L3 is expected to generate fragments of 200,355, 348, 289, and 84 base pairs, while a Sau 3A I digest of H2.16 wouldgenerate a 168 bp fragment in place of the 200 bp fragment. Thisdifference in the Sau 3AI digestion products was used to confirm thatthe three BCA cell lines express at least two different L3 alleles. TheL3 RT-PCR products from all 5 cell lines and thymus RNA were digestedwith Sau 3AI and analyzed on an agarose gel. As shown in FIG. 8B all 3BCA lines express both versions of L3. Remarkably, when this assay wasrepeated using greater amounts of starting material, the 168 bp fragmentwas also detectable in the digests of B/C.N, BCB13 and normal thymuscDNA (Smith et al., unpublished data). To enhance the sensitivity ofthis assay, the PCR was repeated using a P³² end-labeled 5′ L3 specificprimer. The radiolabeled PCR products were digested with Sau3AI andresolved on an agarose gel. As shown in FIG. 8C, B/C.N, BCB13 and thymuscontain the 168 bp fragment. Quantitative analysis indicates that theratio of 200 bp:168 bp fragments in the BCA tumors is 2:1 while theratio of the same fragments detected in B/C.N, BCB13, and thymus isapproximately 20:1. Low levels of expression of this immunogenic L3allele was also observed when RNA from kidney, heart, and skeletalmuscle was analyzed (Smith et al., unpublished data). These resultssuggest that gene deregulation associated with the transformationprocess in the crossreactive tumors leads to the expression of higherlevels of this germ line L3 (C170T) allele, and that this altered L3gene was not generated by somatic mutation of the L3 gene that ispredominantly expressed in normal tissues. The present inventors havetermed this new L3 allele (C170T), the immunogenic L3 allele (iL3).

It is particularly intriguing that the immunogenic L3 allele is alsoexpressed, albeit at a 10 fold reduced level, in normal thymus. Thislevel of expression is evidently not sufficient to tolerize all T cellswith functional avidity for the level of deregulated iL3 expressed insome tumors. The observation that although B/C.N and BCB13 express lowlevels of iL3, they are not susceptible to lysis by the tumor specificCTL suggests, however, that higher affinity T cells have been tolerized.This appears to be the first instance in which a tumor antigen has beenreported to be expressed in the thymus. These observations emphasizethat tolerance to a self-protein is not absolute but must be defined inrelation to quantitative levels of expression (Targoni et al., J. Exp.Med. 187:2055 (1998); C. J. Harrington et al., Immunity 8:571 (1998)).

If broadly effective vaccines are to be developed based on expression ofshared tumor antigens, then it is critical to demonstrate that suchantigens can be immunoprotective. The largest number of shared antigenshave been identified for human tumors, but clinical Immunotherapy trialsemploying these antigens have so far been inconclusive, in part becauseof uncertainty regarding optimal vaccination strategies (Pardoll, D. M.,Nat. Med. 4:525 (1998)). In mice, where immunotherapeutic strategiescould be more thoroughly investigated, very few shared tumor antigenshave been identified. It was, therefore, of considerable interest todetermine whether immunization with iL3 recombinant vaccinia virus wouldinduce tumor specific CTL and protect mice from tumor challenge(Overwijk et al., Proc. Natl. Acad. Sci. 96:2982 (1999); Moss, B.,Science 252:1662 (1991); Irvine et al., J. Immunology 154:4651 (1995);McCabe et al., Cancer Research 55:1741 (1995); Estin et al., Proc. Natl.Acad. Sci. 85:1052 (1988); J. Kantor et al., JNCI 84:1084 (1992); V.Bronte et al., Proc. Natl. Acad. Sci. 94:3183 (1997)). Immunization ofBalb/c mice with vaccinia virus recombinant for the iL3 gene (H2.16)generated CTL that were able to lyse both BCA 34 and BCA 39 tumor cells,but not B/C.N in vitro (FIG. 9A). Mice immunized twice or even once withvaccinia virus recombinant for iL3 were able to reject challenge withBCA 34 tumor cells (FIGS. 9B and 9C). Mice immunized with empty viralvector, or control vaccinia recombinant for the Inhibitor Protein ofcAMP-dependent Protein Kinase (PKIa) were unable to reject this tumorchallenge (Olsen, S. R. and Uhler, M. D., J. Biol. Chem. 266:11158(1991); Mueller et al., Manuscript in Preparation). These resultsdemonstrate that the iL3 self-protein is an immunoprotective tumorantigen.

The present inventors have developed a new strategy to identify genesthat encode CTL epitopes based on CTL-mediated selection from a tumorcDNA library in a modified vaccinia virus vector (Merchlinsky et al.,Virology 238:444 (1997); E. Smith et al., manuscript in preparation). Wehave applied this strategy to identify a deregulated housekeeping genethat encodes a tumor rejection antigen shared by three independentlyderived murine tumors. This ribosomal protein may be representative of alarger class of immunoprotective shared tumor antigens that becomeimmunogenic as a result of deregulated expression of self-proteinswithout compromising immune tolerance to normal tissues. Such antigenswould be well suited for immunotherapy of cancer in vital organs.

Example 5 Expression and Immunogenicity of C35 Tumor Antigen

RNA transcripts of the novel C35 tumor gene are overexpressed in 70%(12/17) of primary human breast carcinomas examined and 50% (5/10) ofbladder carcinomas examined when compared to expression in normal humantissues. The full-length gene encodes a novel 115 amino acid protein ofunknown function. A monoclonal antibody, 2C3, has been selected thatstains the surface membrane of cells expressing C35 by flow cytometricanalysis. In addition, human cytotoxic T lymphocytes (CTL) have beengenerated in vitro that specifically lyse C35+ breast and bladdertumors. The ability to generate C35-specific CTL in vitro from normalhuman donors suggests the absence of tolerance to the overexpressedprotein. Overexpression of C35 in tumors of different individuals andthe ability to induce humoral and cellular immune responses make C35 apromising candidate for immunotherapy.

Material and Methods

Cell lines: Human mammary carcinoma cell lines BT20, BT474, MCF7,MDA-MB231, SKBR3, T47D (supplied by ATCC) were grown in RPMI-1640(BioWhitaker, Walkersville, Md.) supplemented with 10% fetal bovineserum (BIOFLUIDS®, Rockville, Md.). An immortalized line derived fromnormal breast epithelium, H16N2, two metastastic tumors, 21-MT1 and21-MT2, and two primary tumors, 21-NT and 21-PT all derived from thesame patient, and grown in DFCI medium (Band, V. and Sager, R., “TumorProgression in Breast Cancer” in Neoplastic Transformation in Human CellCulture, J. S. Rhim and A. Dritschilo eds., The Human Press Inc.,Totowa, N.J. (1991), pp. 169-78) were generously provided by Dr. VimlaBand, New England-Tufts Medical Center. The bladder tumor cell lineppT11A3 was derived from the immortalized nontumorigenic cell lineSV-HUC. These bladder cell lines were generously provided by Dr.Catherine Reznikoff, University of Wisconsin Clinical Cancer Center, andgrown in F12 medium supplemented with 1% FBS, 0.025 units insulin, 1 ughydrocortisone, 5 ug transferrin, 2.7 g dextrose, 0.1 uM non-essentialamino acids, 2 mM L-glutamine, 100 units penicillin, and 100 ugstreptomycin per 500 ml. Normal proliferating breast epithelial cells(MEC) were purchased from CLONETICS® (BioWhittaker) and maintainedaccording to the supplier's directions.

RNA extraction and Northern Blot Analysis: Cell lines were harvested forRNA extraction at approximately 80% confluency. Cells were harvested andlysed in QG buffer from Qiagen RNAEASY™ kit. Total RNA was extracted asper manufacturer's protocol and stored at −80° C. as precipitates withGITC and alcohol. Tissue samples were provided by the Cooperative HumanTissue Network as snap frozen samples, which were homogenized in lysisbuffer for use in the RNAEASY™ protocol. For Northern blots, mRNA wasextracted from total RNA (30 ug total RNA/well) using Dynal's (LakeSuccess, N.Y.) oligo-dT₂₅ magnetic beads and electrophoresed in 0.8%SEAKEM™ LE (FMC Bioproducts) with 3% formaldehyde. The mRNA was blottedonto GENESCREEN PLUS™ (NEN) in 10×SSC overnight by capillary blot, thenbaked for 2 hours at 80° C. Membranes were probed with random-primed³²P-labeled cDNA probes (PRIMEIT™, Stratagene, LaJolla, Calif.) at 10⁶cpM/ml QUICKHYB™ solution (Stratagene), at 68° C. as per manufacturer'sprotocol. Blots were exposed to Xray film and/or phosphorimager screensovernight. Expression on all blots was normalized to a housekeepinggene, such as GAPDH or beta actin.

Subtractive Hybridization: PCR Select cDNA Subtraction Kit (Clontech,Palo Alto, Calif.), based on Representational Difference Analysis asfirst described by Lisitsyn et al. (Lisitsyn, N. and Wigter, N. M.,Science 259:946-51 (1993)), was employed as per manufacturer's protocolto generate cDNAs enriched for genes overexpressed in tumor compared tonormal breast cell lines. Briefly, oligo-dT-primed double stranded cDNAwas synthesized from 2 ug high quality, DNase-treated mRNA from tumorand normal cells. Adaptors were ligated to short blunt-end (Rsa1digested) tumor sequences and hybridized with excess Rsa1 digestednormal fragments. Following 32 hour hybridization, suppression PCR(Clonte ch) allowed preferential amplification of overexpressed tumorsequences using adaptor sequences as primers. The products of the PCRamplification were cloned into pT7Blue3 (Novagen, Madison, Wis.) togenerate a subtracted library. Clones were grown in LB/ampicillin (10ug/ml) in 96-well format, inserts were PCR amplified from the overnightcultures and PCR products were spotted on GENESCREEN PLUS™ using BioDotmanifold (BioRad, Hercules, Calif.). Duplicate dot blots were thenprobed with random-primed tumor or normal cDNA, or, alternatively, thePCR products of the forward and reverse subtractive hybridizations.Clones that appeared to be overexpressed in the tumor cDNA and forwardsubtraction (tumor minus normal) were analyzed by Northern Blot (asdescribed above) to confirm differential gene expression.

cDNA library and full length gene: Oligo-dT primed double stranded cDNAwas generated from SKBR3 cell line using SMART™ cDNA Synthesis (ClontechLaboratories), followed by phenol:chloroform:isoamyl alcohol extraction.Primers were synthesized (C35 sense: 5′-GCGATGACGGGGGAGCC (SEQ IDNO:2126), and C35 antisense: 5′-CCACGGAATCTTCTATTCTTTCT; (SEQ IDNO:2127; Fisher Oligos, The Woodlands, Tex.) to amplify the codingregion of C35, based on the open reading frame deduced from ESThomologies, Accession #W57569, in particular. PCR products were clonedinto pT7Blue 3 (Novagen).

Vaccinia and Retroviral C35 recombinants: The coding sequence of C35 wassubcloned from the library into vaccinia transfer plasmid, pVTK0 atBamHI/SalI sites in a defined orientation. Recombinant virus wasgenerated by transfection of pVTK0.C35 along with NotI and ApaI digestedV7.5/TK viral DNA into fowlpox virus infected BSC-1 cells. As describedelsewhere (U.S. Utility patent application Ser. No. 08/935,377, now U.S.Pat. No. 6,872,518; PCT/US98/24029; T Cells Specific for Target Antigensand Vaccines Based Thereon), this is an efficient method forconstruction of vaccinia virus recombinants. The C35 gene was alsocloned into a retroviral vector pLXSN, and viral stocks were generatedby co-transfection of 293-GP cells with pVSVg for pseudotyping.Supernatants including infectious virus were collected 48 hours later.

Generation of C35-specific 2C3 monoclonal antibody and FACS analysis:Line1 mouse small cell lung carcinoma cells were infected withC35-retrovirus, and 10³-2×10⁴ cells were injected into three BALB/cByJmice. Following 21 days, serum was harvested from retro-orbital bleedsand checked for reactivity with human tumor cells known to express low(MDA-MB-231) or very high (21NT) levels of C35 mRNA. Spleens were alsoharvested for the production of hybridomas by the fusion of spleen cellswith P3 myeloma cells using standard mouse hybridoma technology. ELISAwas used to screen HAT resistant clones for the presence of Ig. Highproducers were isotyped, quantitated, and used to screen C35+ and C35−cell lines by flow cytometry. Hybridoma clone supernatants containing 1ug IgG were incubated with 10⁶ cells in PAB (PBS, 1% BSA, 0.1% azide)for 30 min on ice, followed by 3 washes with PAB, and incubation withgoat anti-mouse IgG conjugated to FITC (Southern Biotechnology,Birmingham, Ala.) for 30 minutes on ice. One hybridoma clone, 2C3,recapitulated the surface staining seen with the immune serum (FIGS.14A-14B) and was selected for expansion and antibody purification(BioExpress, West Lebanon, N.Y.).

Generation of human C35-specific T cell line: Peripheral blood derivedfrom a healthy female donor (HLA A2, 11, B35, 44) was separated intoerythrocyte-rosette positive fraction (a source of total T lymphocytes)and negative fraction (a source of monocytes). The T lymphocytes werecryopreserved for later use while the monocytes were incubated underconditions to generate dendritic cells (DC). Maturation of DCs wasinduced as described by Bhardwaj and colleagues (Bender, A. et al., J.Immunol. Meth. 196:121-35 (1996); Reddy, A. et al., Blood 90:3640-46(1997); Engelmayer, J. et al., J. Immunology 163:6762-68 (1999)) withsome modifications. hGM-CSF and hIL-4 (1000 U/ml) were added every otherday. At day 7, non-adherent, immature DC were incubated with aretrovirus recombinant for C35 for 6 hours in the presence of GM-CSF andIL-4. At this point, the retroviral supernatant was washed out andimmature dendritic cells were subjected to maturation conditions, whichagain included GM-CSF, IL-4 as well as 12.5% monocyte conditioned medium(MCM). After 4 days, these mature, C35-expressing DC were used tostimulate autologous T cells at a ratio of 1 DC:50 T cells for a periodof 14 days. A fresh pool of autologous DC were generated forrestimulation of the T cells, but this time they were infected after 48hours of maturation in MCM with a vaccinia virus recombinant for C35.Cytokines IL-2 (20 U/ml), IL-12 (20 U/ml) and IL-18 (10 ng/ml) wereadded and a 1:50 ratio of DC:T cells was maintained. Following 12 daysculture, T cells were stimulated for 7 additional days with EBV-B cellsinfected with C35 recombinant retrovirus and with addition of IL-2 (20U/ml) and IL-7 (10 ng/ml). Cytokines were all purchased from R&D Systems(Minneapolis, Minn.). At this point, the cells were >90% CD8⁺ and weretested for activity in a standard ⁵¹Cr Release assay. Briefly, onemillion target cells were incubated with 100 uCi ⁵¹Cr, washed, thenincubated with CTL effectors for 4 hours in RPMI-1640, supplemented with10% human AB serum (BioWhittaker). Activity of the CTL is expressed asthe percent of specific lysis, measured as (⁵¹Cr released into thesupernatant upon lysis of labeled targets by CTL−spontaneousrelease)/(maximal release−spontaneous release).

Results

Characterization of C35: The sequence of clone C35, differentiallyexpressed in human breast tumor cells, is not homologous to any knowngene in Genbank, but homologous EST sequences (prototype Accession#W57569) were identified. Homologous human EST fragments are present inNCI CGAP (Cancer Genome Anatomy Project) libraries, including tumors ofbrain, lung and kidney (A# AA954696), Soares ovary (A# AA285089) andparathyroid tumors (A# W37432), an endometrial tumor (A#AA337071), andcolon carcinoma (A# AA313422). An open reading frame was identified thatencodes a 115 amino acid protein (FIG. 10A). The full-length gene wasisolated from a cDNA library of the breast adenocarcinoma cell lineSKBR3. Sequencing of full-length transcripts from the cell lines SKBR3,21MT2-D, and H16N2 confirmed that there were no point mutations in thecDNA; the transcript is 100% homologous in C35^(hi) cell lines, as wellas C35^(lo) cell lines. The C35 gene aligns on human chromosome 17q12(A# AC040933) and mouse chromosome 11 (A# AC064803). Exons were deducedfrom homologies with cDNA EST sequences, as well as GRAIL predictions.Interestingly, the gene for C35 is within 1000 base pairs of theHer2/neu oncogene and within 2000 bp of the gene for Growth FactorReceptor-Bound Protein 7 (GRB7), a tyrosine kinase that is involved inactivating the cell cycle and that is overexpressed in esophagealcarcinomas (Tanaka, S. et al., J. Clin. Invest. 102:821-27 (1998)) (FIG.10B). Her2/neu protein overexpression has been correlated with geneamplification in 30% breast tumors and is associated with poor clinicalprognosis (Slamon, D. J. et al., Science 235:177-82 (1987)).

Predicted protein motifs in the C35 amino acid sequence include: caseinkinase II phosphorylation sites at amino acids 38 to 41 (TYLE), 76 to 79(SKLE), and 97 to 100 (SNGE); an N-myristoylation site at amino acids 60to 65 (GGTGAF); and a cAMP- and cGMP-dependent protein kinasephosphorylation site at amino acids 94 to 97 (RRAS), all of SEQ ID NO:2.Finally, the C35 protein contains a prenylation motif at theCOOH-terminus, amino acids 112 to 115 of SEQ ID NO:2 (CVIL).Prenylation, the covalent attachment of a hydrophobic isoprenoid moiety,is a post-translational modification that promotes membrane associationand also appears to mediate protein-protein interactions (Fu, H.-W. andCasey, P. J., Recent Progress in Hormone Research 54:315-43 (1999)).Prenylation has been shown to be required for localization andtransforming potential of the oncogenic Ras family proteins to the cellsurface (Jackson, J. H. et al., Proc. Natl. Acad. Sci. U.S.A. 87:3042-46(1990); Hancock, J. F. et al., Cell 57:1167-77 (1989)). Inhibitors ofprenylation have been shown to possess anti-tumor activities, such asslowing tumor growth (Garcia, A. M. et al., J. Biol. Chem. 268:18415-18(1993)) and to promote rejection in animal models (Kohl, N. E. et al.,Nature Med. 1:792-97 (1995)). Three O-glycosylation sites are predictedat or near the amino terminus—thr8, ser2, and ser9 using NetOGlyc2.0.

C35 Transcript is Overexpressed in Breast and Bladder Carcinoma: Anideal target antigen for tumor immunotherapy would be abundantlyexpressed in multiple independent carcinomas, and would be absent orminimally expressed in normal proliferating and vital tissues.Differential expression of C35 was confirmed by Northern blot analysis.C35 is expressed in 7/10 human tumor cell lines at levels 10-25× higherthan expression in a normal immortalized breast epithelial cell line,H16N2 (FIG. 11A). Importantly, C35 expression is shared among linesderived from both primary (21NT, 21PT) and metastatic (21MT1, 21MT2)lesions of a single patient, suggesting its expression may be associatedwith early events in the process of tumor transformation. In addition,the overexpression of C35 is shared among independently derived humanmammary carcinoma cell lines, including SKBR3, T47D, and BT474.Interestingly, the C35 expression pattern in SKBR3, MDA MB231, H16N2 andtumors derived from the same patient correlates with Her2/neuexpression, which may be associated with the close genomic proximity ofthe two genes and the incidence of HER2/neu gene amplification.

To investigate whether C35 expression in patient derived tumors isclinically relevant for development of a cancer vaccine, mRNA wasextracted from snap frozen human tissue samples obtained from theCooperative Human Tissue Network (CHTN). 70% of primary breast tumorsamples overexpress C35 transcript (FIG. 11B), and 35% (7/20) of thesebreast adenocarcinomas overexpress at levels 10-70 fold higher thannormal breast. Overexpression of C35 is also seen in 50% of bladdercarcinoma primary specimens examined (FIG. 12), while 20% (3/14) ofprimary bladder carcinoma express at levels greater than 10 fold higherthan normal bladder. Overexpression of C35, at levels 9× or greater, wasnot detected in panels of ovarian (0/7), prostate (0/5), or colon (0/15)carcinomas (data not shown).

2C3 Monoclonal Antibody reacts with C35+ cells: In order to confirmdifferential expression of the gene product encoded by C35, a monoclonalantibody against the shared tumor antigen was selected. Hybridomas wereproduced by immunizing mice with a poorly immunogenic BALB/cByJ tumorcell line, which had been transduced with a retroviral human C35recombinant. Hybridoma clones were screened for their ability to stainC35++breast and bladder tumor cell lines (FIGS. 13A and 13B).Non-tumorigenic breast H16N2 and bladder SV-HUC epithelial cell linesdid not show a significant shift in fluorescence intensity when comparedto the isotype control. In contrast, 2C3 monoclonal antibodyspecifically stained C35+ breast tumors, SKBR3 and 21-NT-D, and bladdertumor ppT11A3. The staining was carried out on cells that were neitherfixed nor permeabilized, indicating that 2C3 antibody recognizes asurface molecule.

Inhibition of Tumor Growth with C35 Antibodies:

Antibodies are useful tools to detect diagnostic markers of cancer, butthey may also have potential use for therapeutic applications. HumanizedHer2/neu specific antibody (HERCEPTIN™) has been successfully employedfor treatment of some breast cancers. HERCEPTIN™ binds HER2/neu anddownregulates signal transduction from the growth factor receptor.Growth inhibition studies were performed with C35-specific 2C3 antibody.21NT-D breast tumor and H16N2 “normal” breast cell lines were grown invitro in the presence of various antibody concentrations. An XTT assaywas performed to evaluate cell expansion at 72 hours. Results shown inFIGS. 14A and 14B indicate that 2C3 inhibits growth of 21NT tumor cellsby approximately 50% at concentrations as low as 1 ug/ml.

A C35 Class I Epitope is HLA-A2 Restricted:

Establishment of self-tolerance is a major obstacle to development ofvaccines based on self proteins. Tolerance, however, must be defined interms of quantitative levels of expression. It is possible that evenwhile high affinity antigen-specific T cells are tolerized, T cells withlower affinity receptors that do not have functional avidity for a lowconcentration of antigen escape tolerance induction. These same T cellscould, however, subsequently become functionally significant if there ismarkedly increased avidity associated with overexpression of the targetantigen. Even if they are few in number, such T cells could be expandedby the most fundamental of immunological manipulations, vaccination.

C35 is a self-protein expressed at low basal levels in normal humantissues. It was, therefore, necessary to determine if human T cells aretolerant to C35 at levels of expression characteristic of carcinomas.The only way to exclude tolerance is by demonstrating responsiveness,and the only way to demonstrate responsiveness short of a clinical trialis by in vitro stimulation. Human T cells and autologous dendritic cellswere derived from PBL from a normal donor. The T cells were primed byalternate stimulation with autologous dendritic cells infected withretroviral or pox virus recombinants of the C35 cDNA. CTL recovered invitro following several cycles of stimulation were analyzed for theirability to lyse C35+ target tumor cells (FIGS. 15A and 15B) or tosecrete cytokines in response to antigen induced activation (FIGS. 16Aand 16B). The targets either endogenously expressed C35 and/or HLA-A2.1,or were engineered to express these proteins via standard transfectionwith a C35-recombinant mammalian expression vector, or by infection withC35-recombinant vaccinia virus. Previous studies have demonstrated thatprotein expression by vaccinia virus is an efficient means of targetingpeptides to the MHC-I processing pathway (Moss, B., Science 252:1662-67(1991).

Following several rounds of stimulation, both a bulk T cell line and a Tcell clone were selected that differentially lyse C35+ tumor cellsrelative to C35^(lo) H16N2 normal breast epithelial cell line in a ⁵¹Crrelease assay (FIGS. 15A and B). The HLA-A2 restricted C35-specific CTLclone 10G3 efficiently lysed the HLA-A2 transfected tumorigenic cellline, 21-NT.A2, which expresses C35 antigen at levels 15× greater thanH16N2 and is stained with 2C3 monoclonal antibody. Specific lysis wasalso with the HLA-A2+ bladder tumor cell line ppT11A3 compared to thenon-tumorigenic bladder cell line SV-HUC from which it was derived (FIG.15B). The data demonstrate CTL sensitivity of tumors that express highlevels of C35 with minimal lysis of C3511 nontumorigenic immortalizedcell lines. Importantly, the same CTL are not reactive with MEC, aprimary culture of non-immortalized, non-transformed, HLA-A2⁺ breastepithelial cells that do not express C35 at significant levels. Furtherevidence to support C35+ tumor recognition by the T cells is shown inFIGS. 6A and 16B. The T cells secrete IFN-gamma and TNF-alpha inresponse to C35+, HLA-A2+ stimulator. Again, the non-tumorigenic,C35^(lo) cell line H16N2.A2 did not induce cytokine secretion byC35-specific T cells. However, infection of this line with vacciniavirus recombinant for C35 confers the ability to activate the T cells.Since the T cells do not secrete IFN-gamma or TNF-alpha in response toH16N2.A2 transduced with an irrelevant protein L3, this indicates thatthe response is specific to C35 protein expression (FIGS. 16A and B).

Following several rounds of stimulation, both a bulk T cell line and a Tcell clone were selected that differentially lyse C35+, HLA-A2+ tumorcells in a ⁵¹Cr release assay. The C35-specific CTL did not lyse theHLA-A2 transfected non-tumorigenic breast epithelial cell line, H16N2.A2(FIGS. 15A and 15B), although this cell line does express C35 at lowlevels based on the Northern blot data shown in FIG. 11A. However,C35-specific CTL efficiently lysed the HLA-A2 transfected tumorigeniccell line, 21-NT.A2, which expresses C35 antigen at levels 15× greaterthan H16N2 and is stained with 2C3 monoclonal antibody. C35⁺tumor-specific lysis was also shown with the bladder tumor cell lineppT11A3 compared to the non-tumorigenic bladder cell line SV-HUC fromwhich it was derived. The data demonstrate CTL sensitivity of tumorsthat express high levels of C35 with minimal lysis of C35^(lo)nontumorigenic immortalized cell lines. Importantly, the same CTL arenot reactive with MEC, a primary culture of non-immortalized,non-transformed, HLA-A2⁺ breast epithelial cells that do not express C35at significant levels. Further evidence to support C35+ tumorrecognition by the T cells is shown in FIGS. 16A and 16B. The T cellssecrete IFN-gamma and TNF-alpha in response to C35+, HLA-A2+ stimulator.Again, the non-tumorigenic, C35^(lo) cell line H16N2.A2 did not inducecytokine secretion by C35-specific T cells. However, infection of thisline with vaccinia virus recombinant for C35 confers the ability toactivate the T cells. Since the T cells do not secrete IFN-gamma orTNF-alpha in response to H16N2.A2 transduced with an irrelevant proteinL3, this indicates that the response is specific to C35 proteinexpression.

The C35-specific T cells were generated from a donor with HLA haplotypeA2, A11, B8, B35. The bladder cell lines, SV-HUC and ppT11A3 derive froma donor with haplotype HLA-A2, B18, B44. However, since the H16N2immortalized breast epithelial cell line and 21-NT and 21-MT breasttumor cell lines derived from the same HLA-A2 negative donor, these celllines had to be transfected with HLA-A2.1 to provide a required MHCrestriction element for recognition by HLA-A2 restricted 10G3 T cellclone (FIGS. 16A and 16B). The T cells were strongly stimulated tosecrete these lymphokines by the breast lines that expressed both C35and HLA-A2 (compare 21-MT2 with 21-MT2.vvA2). The data indicate thatthere is at least one HLA-A2.1 defined epitope of C35.

Deletion mutants of C35 coding region were constructed to identify cDNAsegments that encode the peptide epitope recognized by the CTL. FIGS.15A and 15B demonstrate almost equivalent IFN-gamma and TNF-alphasecretion by T cells stimulated with the full length C35 or a truncatedmutant encompassing only the first 50 amino acids.

Discussion

C35 is a novel tumor antigen that is overexpressed in breast and bladdercarcinoma. The gene has properties that make it a promising candidatefor tumor immunotherapy. It is expressed in a significant number oftumors derived from different individuals. Expression in vital normaltissues is relatively low, reducing the risk of autoimmune reactionsand, equally important, making it unlikely that immune cells have beenrendered tolerant to the gene product. C35 is characterized as a “tumorantigen” since C35 expressing dendritic cells induce autologous tumorspecific human cytotoxic T lymphocytes in vitro.

C35 is a novel gene product of unknown function. However, our studieswith monoclonal antibodies have provided some insight into thelocalization of the protein. Both serum and a monoclonal antibodyderived from a C35-immunized mouse specifically stain unfixed cells thatexpress C35. This suggests that the antibody recognizes a tumor surfacemembrane protein. Although the protein sequence does not conform withknown transmembrane motifs based on hydrophobicity, the existence of aprenylation site at the COOH terminus suggests insertion into themembrane. Prenylation is a post-translational lipid modification thatproduces a substantially more hydrophobic protein with high affinity forthe membrane (Fu, H.-W. and Casey, P. J., Recent Progress in HormoneResearch 54:315-43 (1999)). Other proteins that contain prenylationsites include the Ras oncogene family. Ras GTPases act in signaltransduction cascades with MAPK to induce cell division andproliferation. Ras proteins are anchored to the plasma membrane viaprenylation, but the proteins remain in the cytoplasmic face of themembrane. Therefore, it is possible that C35 also remains on thecytoplasmic side of the membrane, but there may be sufficient transportto the outer surface to be detected with a specific antibody.

C35-specific antibodies are valuable tools for studying the proteinexpression of C35, to corroborate Northern blot analysis, and for use inassays such as Western blots and immunohistochemistry. In addition,these antibodies may have therapeutic benefits, such as has beenrecently been demonstrated for HERCEPTIN™ (Baselga, J. et al., J. Clin.Oncol. 14:737-44 (1996); Pegram, M. D. et al., J. Clin. Oncol.16:2659-71 (1998)), an antibody to the tumor-associated antigen HER2-neu(c-erbB-2) (Schechter, A. L. et al., Nature 312:513-16 (1984).HERCEPTIN™'s anti-tumor effects include binding the epidermal growthfactor receptor, which inhibits tumor cell growth, and elicitingantibody dependent cell-mediated cytotoxicity (Dillman, R. O., CancerBiotherapy & Radiopharmaceuticals 14:5-10 (1999).

Example 6 Induction of Cytotoxic T Cells Specific for Target Antigens ofTumors

Human tumorspecific T cells have been induced in vitro by stimulation ofPBL with autologous tumors or autologous antigen presenting cells pulsedwith tumor lysates (van Der Bruggen, P. et al., Science 254: 16431647(1991); Yasumura, S. et al., Cancer Res. 53: 146168 (1993); Yasumura, S.et al., Int. J. Cancer 57: 297305 (1994); Simons, J. W. et al., CancerRes. 57: 153746 (1997); Jacob, L. et al., Int. J. Cancer 71:325332(1997); Chaux, P. et al., J. Immunol. 163:29282936 (1999)). PBL havebeen derived from either patients deliberately immunized with tumor,with tumor modified to enhance its immunogenicity, or with tumorextracts, or patients whose only prior stimulation was in the naturalcourse of disease. T cells with reactivity for infectious agents couldbe similarly derived by in vitro stimulation of T cells with autologouscells that have been either infected in vitro or were infected in vivoduring the natural course of exposure to the infectious agent. CD4+ andCD8+ T cells or antibody selected under these or other conditions to bespecific for either tumor cells or cells infected with either a virus,fungus or mycobacteria or T cells or antibodies specific for the targetantigens of an autoimmune disease could be employed in the selection andscreening methods of this invention to detect and isolate cDNA thatencode these target antigens and that have been incorporated into arepresentative cDNA library.

In spite of demonstrated success in the induction of human T cellresponses in vitro against a number of antigens of tumors and infectedcells, it is not certain that these represent the full repertoire ofresponses that might be induced in vivo. Because safety considerationslimit the possibilities of experimental immunization in people, there isa need for an alternative animal model to explore immune responses tohuman disease antigens. The major obstacle to developing such a model isthat numerous molecules expressed in normal human cells are stronglyimmunogenic in other species. It is, therefore, necessary to devise ameans of inducing tolerance to normal human antigens in another speciesin order to reveal immune responses to any human diseasespecificantigens. It is now recognized that activation of antigenspecific Tlymphocytes requires two signals of which one involves presentation of aspecific antigenic complex to the T cell antigen receptor and the secondis an independent costimulator signal commonly mediated by interactionof the B7 family of molecules on the surface of the antigen presentingcell with the CD28 molecule on the T cell membrane. Delivery of anantigenspecific signal in the absence of a costimulator signal not onlyfails to induce T cell immunity but results in T cell unresponsivenessto subsequent stimulation (Lenschow, D. J. et al., Ann. Rev. Immunol.14:233258 (1996)). Additional studies have revealed a key role foranother pair of interactions between the CD40 molecule on the antigenpresenting cell and CD40 ligand on the T cell. This interaction resultsin upregulation of the B7 costimulator molecules (Roy, M. et al., Eur.J. Immunol. 25:596603 (1995)). In the presence of antiCD40 ligandantibody either in vivo or in vitro, the interaction with CD40 isblocked, B7 costimulator is not up regulated, and stimulation with aspecific antigenic complex results in T cell tolerance rather than Tcell immunity (Bluestone, J. A. et al., Immunol. Rev. 165:512 (1998)).Various protocols to block either or both CD40/CD40 ligand interactionsand B7/CD28 interactions have been shown to effectively inducetransplantation tolerance (Larsen, C. et al., Nature 381:434-438 (1996);Kirk et al., Nature Medicine 5:686693 (1999)). An example of the effectof antiCD40 ligand antibody (antiCD154) in blocking the reactivity ofmurine T cells to specific transplantation antigens is shown in FIGS.17A and 17B. DBA/2 (H2^(d)) mice were immunized with 10⁷ C57Bl/6(H2^(b)) spleen cells intraperitoneally and, in addition, were injectedwith either saline or 0.5 mg monoclonal antiCD40 ligand antibody (MR1,antiCD154, Pharmingen 09021D) administered both at the time ofimmunization and two days later. On day 10 following immunization,spleen cells from these mice were removed and stimulated in vitro witheither C57Bl/6 or control allogeneic C3H(H2^(k)) spleen cells that hadbeen irradiated (20 Gy). After 5 days in vitro stimulation, C57Bl/6 andC3H specific cytolytic responses were assayed at various effector:targetratios by ⁵¹Cr release assay from specific labeled targets, in thiscase, either C3H or C57Bl/6 dendritic cells pulsed with syngeneic spleencell lysates. The results in FIGS. 17A and 17B show that significantcytotoxicity was induced against the control C3H alloantigens in bothsaline and antiCD154 treated mice whereas a cytotoxic response toC57Bl/6 was induced in the saline treated mice but not the antiCD154treated mice. This demonstrates specific tolerance induction to theantigen employed for immune stimulation at the time CD40/CD40 ligandinteractions were blocked by antiCD154.

A tolerization protocol similar to the above employing either antiCD154alone or a combination of antiCD154 and antiB7 or antiCD28 could beemployed to induce tolerance to normal human xenoantigens in mice priorto immunization with a human tumor. In one embodiment, the normalantigens would be expressed on immortalized normal cells derived fromthe same individual and tissue from which a tumor cell line is derived.In another embodiment, the normal and tumor antigens would derive fromcell lysates of normal and tumor tissue of the same individual eachlysate pulsed onto antigen presenting cells for presentation tosyngeneic murine T cells both in vivo and in vitro. In a preferredembodiment, the tumors would derive by in vitro mutagenesis or oncogenetransformation from an immortalized, contactinhibited,anchoragedependent, nontumorigenic cell line so that very wellmatchednontumorigenic cells would be available for tolerance induction.

An alternative to the tolerization protocols is depletion of T cellsthat are activated by normal antigens prior to immunization with tumor.Activated T cells transiently express CD69 and CD25 with peak expressionbetween 24 and 48 hours poststimulation. T cells expressing thesemarkers following activation with normal cells or normal cell lysatescan be depleted with antiCD69 and antiCD25 antibody coupled directly orindirectly to a matrix such as magnetic beads. Subsequent immunizationof the remaining T cells with tumor cells or tumor cell lysates eitherin vitro or in vivo following adoptive transfer will preferentially giverise to a tumorspecific response.

In one embodiment, the mice to be tolerized to normal human cells orlysates and subsequently immunized with tumor cells or lysates are anyof a variety of commercially available inbred and outbred strains.Because murine T cells are restricted to recognize peptide antigens inassociation with murine MHC molecules which are not expressed by humancells, effective tolerization or stimulation requires eithertransfection of human cells with murine MHC molecules or representationof human normal and tumor antigens by mouse antigen presenting cells.Dendritic cells are especially preferred as antigen presenting cellsbecause of their ability to represent antigenic peptides in both theclass I and class II MHC pathways (Huang, et al., Science 264:961965(1994); Inaba, et al., J. Exp. Med. 176:1702 (1992); Inaba, et al., J.Exp. Med. 178:479-488 (1993)). In another embodiment, mice doubletransgenic for human HLA and human CD8 or CD4 are employed. The HLAtransgene permits selection of a high affinity, HLArestricted T cellrepertoire in the mouse thymus. In addition, a human CD8 or CD4transgene is required because murine CD8 and CD4 do not interactefficiently with the cognate human class I or class II MHC molecules.The use of nontransgenic mice to generate human tumorspecific T cellswould lead to identification of any human tumor antigens that can beprocessed in association with murine MHC molecules. Since multiplemurine strains with diverse MHC molecules are available, this couldencompass a wide range of antigens. However, it would have to beseparately determined by stimulation of human T cells with autologousantigen presenting cells whether these tumorspecific antigens alsoexpress peptides that can be processed and presented in association withhuman HLA. Such peptides may or may not overlap with those initiallydetected in association with murine MHC molecules but would derive fromthe same set of proteins. By employing HLA transgenic mice it ispossible to more directly address the relevance of antigenic peptides tohuman MHC. There can, however, be no assurance that peptide processingwill be identical in murine and human antigen presenting cells. It isessential, therefore, to confirm that HLArestricted, human tumorantigenspecific T cells are indeed also crossreactive on human tumorcells. Finally, no matter how the issue of processing and presentationin association with human HLA is addressed, it must in all cases bedetermined whether human T cells are reactive to the identified antigensor whether they have been rendered tolerant, perhaps due to expressionof the same or a related antigen in some other nonhomologous normaltissue. Relevant information on this point can be obtained through invitro stimulation of human T cell responses with the identified antigensor antigenic peptides presented by autologous antigen presenting cells.Ideally, it would be shown that patients with antigen positive tumorshave an increased frequency of T cells reactive with the purportedtumorspecific antigen. To demonstrate that the antigenspecific human Tcells induced can be effective in eradicating tumors, the selected humanT cells could be adoptively transferred into SCID mice bearing a humantumor xenograft as described by Renner, C. et al., Science 264:833835(1994). However, definitive evidence for clinical relevance would awaitthe results of a human clinical trial.

Conditions for in vitro stimulation of primary human T cell responsesare described in Example 2 and are applicable to both CD4+ and CD8+responses. The strategies described for induction of human T cell orantibody responses specific for human tumors are equally applicable toinduction of T cell or antibody responses to target antigens of humancells infected with either a virus, fungus or mycobacteria. Indeed, inthis case the same uninfected cell population affords an immediatelyavailable normal control population for tolerance induction and toconfirm infectious specificity.

The construction of transgenic mice is well known in the art and isdescribed, for example, in Manipulating the Mouse Embroy: A laboratoryManual, Hogan, et al., Cold Spring Harbor Press, second edition (1994).Human CD8 transgenic mice may be constructed by the method of LaFace, etal., J. Exp. Med. 182:131525 (1995). Construction of new lines oftransgenic mice expressing the human CD8alpha and CD8beta subunits maybe made by insertion of the corresponding human cDNA into a human CD2minigene based vector for T cellspecific expression in transgenic mice(Zhumabekov, et al., J. Immunol. Methods 185:133140 (1995)). HLA class Itransgenic mice may be constructed by the methods of Chamberlain, etal., Proc. Natl. Acad. Sci. USA 85:76907694 (1988) or Bernhard, et al.,J. Exp. Med. 168:11571162 (1988) or Vitiello, et al., J. Exp. Med.173:10071015 (1991) or Barra, et al., J. Immunol. 150:36813689 (1993).

Construction of additional HLA class I transgenic mice may be achievedby construction of an H2Kb cassette that includes 2 kb of upstreamregulatory region together with the first two introns previouslyimplicated in gene regulation (Kralova, et al., 1992, EMBO J. 11:45914600). Endogenous translational start sites are eliminated from thisregion and restriction sites for insertion of HLA cDNA are introducedinto the third exon followed by a polyA addition site. By including anadditional 3 kb of genomic H2Kb sequence at the 3′ end of thisconstruct, the class I gene can be targeted for homologous recombinationat the H2Kb locus in embryonic stem cells. This has the advantage thatthe transgene is likely to be expressed at a defined locus known to becompatible with murine class I expression and that these mice are likelyto be deficient for possible competition by H2Kb expression at the cellmembrane. It is believed that this will give relatively reproducibleexpression of diverse human HLA class I cDNA introduced in the sameconstruct.

Example 7 Induction of GM-CSF Secretion by Line 4 T Cells Stimulatedwith the C35 Peptide Epitopes

T cell line 4 was generated by stimulating normal donor T cells for 12days each with autologous dendritic cells (DC) and then autologousmonocytes infected with C35 recombinant vaccinia.virus. Weeklystimulation was continued with allo PBL and the 21NT tumor transfectedwith HLA-A2/Kb (21NT-A2). The results of the experiment are depicted inFIG. 18. For this experiment, T cells were restimulated in vitro at 10⁶T cells per well with 5×10⁴ irradiated (2500 rads) H16N2-A2/Kb pulsedwith 1 ug/ml of C35 peptides 9-17, 77-85, 104-112, or 105-113 and 105irradiated allo PBL per well with IL2 (20 U/ml) and IL-7 (10 ng/ml) inAIM-V/5% human AB serum. After two (2) rounds of stimulation for 7 days,T cells were tested for induction of GM-CSF secretion followingincubation with different stimulators pulsed or not pulsed with 1 ug/mlof peptide or infected with vvC35 or vvWT at MOI=1. T cells (5000) wereincubated with 25000 of the various stimulator cells overnight inAIM-V/5% human AB serum in triplicate.

As shown in FIG. 18, no GM-CSF stimulation was observed with normalbreast epithelial cells transfected with HLA-A2/Kb (H16N2-A2). However,the same cells pulsed with the C35 peptides produced significantstimulation (A16N2-A2+peptide). Stimulation was seen for cells pulsedwith each peptide, with peptide 77-85 showing the greatest stimulation.In addition, significant stimulation was seen with the 21NT tumor cellstransfected with HLA-A2/Kb (21NT-A2) but not pulsed with C35 peptides,as well as normal breast epithelial cells transfected with HLA-A2/Kb andinfected with C35 recombinant vaccinia virus (H16N2-A2 vvC35). Asexpected, no stimulation was seen with normal breast epithelial cellstransected with HLA-A2/Kb and infected with wild-type vaccinia virus.

The fact that minimal stimulation was seen H16N2-A2 cells that were notpulsed with any of the C35 peptides yet substantial stimulation was seenwith the same cells pulsed with peptides confirms that each of the C35peptides tested associates with HLA2. Moreover, the fact that the21NT-A2 tumor cells also showed stimulation even though they had notbeen pulsed with any C35 peptides confirms that the C35 peptides areproduced (i.e., processed) by the tumor cells. Finally, the fact thatstimulation was observed with the H16N2-A2 cells infected with C35recombinant vaccinia virus confirms that the full-length C35 polypeptideintroduced recombinantly is processed in the cells.

Example 8 Generation of Peptide Specific CD8⁺ T Cells

CD8⁺ T Cell Selection

PBMCs were harvested using standard Ficoll-Hypaque separation ofanti-coagulated human blood. Whole blood was diluted with HBSS (w/oCa²⁺/Mg²⁺) 2:1, in 50 ml centrifuge tubes. 30 ml of blood was thenlayered over 12 ml ficoll. The blood was centrifuged at 18° C., 400×gfor 30 minutes, with the brake off. The interface layer containing PBMCwas removed and washed 2× with HBSS.

CD8 positive cells were selected via magnetic activated cell sorting(MACS) manufactured by Miltenyi Biotec, Auburn, Calif. Specifically, thePBMCs are pelleted and resuspended in 80 μl MACS buffer (degassed,ice-cold PBS pH 7.2, supplemented with 0.5% BSA and 2 mM EDTA) per 10⁷total cells. 20 μl of MACS CD8 microbeads was added per 10⁷ cells, mixedwell and incubated for 15 minutes at 6-12° C. The cells were then washedby adding 12 ml MACS buffer, centrifuged at 300×g for 10 minutes at 4°C., the supernatant removed completely and the pellet resuspended in 1-2ml MACS buffer. A positive selection VS⁺ MACS column was placed in themagnetic field of a separator. The column was prepared by washing with 3ml MACS buffer. The cell suspension was applied to the column, allowingthe cells to completely enter the column. The column was then rinsedwith 3×3 ml MACS buffer.

The column was removed from the separator and placed in a collectiontube. 5 ml of MACS buffer was then pipetted onto the column and thecells firmly flushed through with supplied plunger. The CD8⁺ cell typewas verified via flow cytometry. The cells were either kept in culturein AIM-V media (Gibco, Carlsbad, Calif.) with 10% human AB serum (GeminiBioproducts, Woodland, Calif.) or frozen in 10% DMSO & 90% human ABserum at −80° C. for later use.

Generation & Maturation of Dendritic Cells (DCs)

DC Selection from PBMCs

The PBMCs were plated out, either inclusive of all PBL or following CD8⁺and/or CD4⁺ cell selection. CD4⁺ cells were selected in the same manneras the CD8⁺ cells (above) using Miltenyi Biotec MACS CD4 microbeads. Theabsence or presence of PBL does not make a difference in DC selection.PBMCs were then plated at 5-7×10⁶ cells/well in 6-well plates in RPMIserum free media for 2 hrs at 37° C. Non-adherent cells were removed andthe adherent layer washed with 3× with HBSS. Adherent cells wereincubated in STEMSPAN™ (StemCell Technologies, Vancouver, BC, Canada)with 1% human AB serum, 1000 U/ml GM-CSF and 1000 U/ml IL-4 at 37° C.,7% CO₂ for 7 days for generation of immature Dcs. Media and cytokineswere replenished every other day.

DC Maturation

On day 7 immature DCs were collected by harvesting the non-adherentcells. The DCs were incubated in STEMSPAN™ with 1% human AB serum, 1000U/ml GM-CSF, 1000 U/ml IL-4, 10 ng/ml TNFα and 1 μg/ml CD40L for 24-48hrs, 37° C., 7% CO₂, to mature.

CD8⁺ Cell Stimulations with Peptide Pulsed APCs

A first stimulation was performed utilizing peptide pulsed autologousDcs. The CD8⁺ cells used were either fresh or brought back into culturefrom −80° a minimum of 24 hr prior to the stimulation. Fresh matured DCswere plated at 1×10⁴ cells per well of 96-well U-bottom plate in AIM-Vmedia. 10 μg/ml peptide and 3 μg/ml β₂-microglobulin were added toindividual wells for a 4 hr pulse, 37° C., 7% CO₂.

Wells were combined for pools of peptide pulsed DCs where indicated.Cells were then transferred to 15 ml conical tubes, and the volumebrought up to 12 ml with AIM-V media. The pellet was centrifuged andresuspended in AIM-V media and irradiated at 2500 rads. On Day 0, 1×10⁵peptide pulsed DCs were added to 2×10⁶ CD8⁺ cells in 24-well plates at aratio of 20 T-cells to 1 DC for stimulations. The cells were maintainedin AIM-V with 5% human AB serum and 10 ng/ml IL-7. On Day 1, 20 IU/mlIL-2 was added to each well. The wells were then incubated at 37° C., 7%CO₂ for 12 days, refreshing media when necessary.

2^(nd) & 3^(rd) Stimulations

The protocol for the second and third stimulations was similar to theprotocol for the 1^(st) stimulation with the following exceptions.Frozen DCs were utilized by bringing back into culture and maturing. TheDCs were pulsed with 1-10 μg/ml peptide (experimentally dependent) for2-4 hr. The DCs were irradiated at 2500 rads, omitting the washing step.Thus, any unbound peptide remained in media. The cells were maintainedat a 20:1 ratio (CD8:DC) in 12 or 24-well plates, plating 1×10⁶ CD8⁺cells/well. The T cells were assayed for target cell recognition viacytotoxicity and/or cytokine release assays 7-12 days post the 3^(rd)stimulation.

4^(th) Stimulation and Beyond

Various cell types can be used as APCs, i.e., DC, monocytes, EBV-B andtumor. Autologous monocytes are given as an example. PBMCs areirradiated at 2500 rads, then plated out at 4-6×10⁶ cells/well of12-well plate in RPMI, and incubated for 2 hr at 37° C., 7% CO₂.Non-adherent cells are removed and the adherent monocytes washed 2× withHBSS.

The monocytes are then pulsed with peptides at 1-10 μg/ml in AIM-V mediafor 2 hours, 37° C., 7% CO₂. Where CD8⁺ cells have previously beenstimulated with peptide pulsed APCs in pools, combine peptides whenpulsing monocytes into those same pools. Add 1-10 μg/ml of each peptide.CD8⁺ cells are added to the respective pulsed monocytes, maintaining the20:1 ratio.

On Day 0 10 ng/ml IL-7 are added, and on Day 1 20 IU/ml IL-2 are added.The cells are then incubate at 37° C., 7% CO₂, for 7 days refreshingmedia when necessary. In general, T cells are assayed for target cellrecognition via cytotoxicity and/or cytokine release assays 5-7 dayspost each stimulation.

Clonal CD8⁺ Cell Populations

Cells were cloned out by limiting dilution. To the wells of 96-wellU-bottom plates were added: 1-10 CD8⁺ cells, 1×10⁴ mixed irradiatedallogeneic feeder cells (2500 rads), 1000 peptide pulsed autologousirradiated EBV-B cells (2500 rads) and 10 ng/ml IL-7 in AIM-V media with5% human AB serum. 20 IU/ml IL-2 was added on Day 1. Clones wererefreshed weekly by changing 50% of the media, adding fresh cytokinesand irradiated feeder and peptide pulsed B cells. Clones were selectedfor target cell recognition assays, cytotoxicity and/or cytokinerelease, and expansion, from plates showing <30% growth, indicatingclonality of the limiting dilution.

Rapid Expansion Protocol

This protocol was utilized for clones showing lytic activity. (Protocolreceived from Dr. Steven Rosenberg (NCI) via personal communication.)

On Day 0, allogeneic mixed PBMC were irradiated (2500 rads) and added toa 25 cm² flask (4×10⁷ cells/flask) in 25 ml AIM-V with 10% human ABserum. OKT3 was added at 30 ng/ml followed by 1×10⁵ viable cloned CD8⁺cells. On Day 2, IL-2 was added at 300 IU/ml. On Day 5, 20 ml mediachanged/flask, 300 IU/ml fresh IL-2 was added. Immune activity wastested for by cytotoxicity or cytokine release assay 8-11 days afterREP.

Cytotoxicity ⁵¹Cr Release Assay

CD8⁺ cells were screened for activity against selected target cells.APCs were pulsed with peptide and cells transduced to express retrovirusor vaccinia virus, tumor, normal controls, etc. Targets were washed byremoving cells from flasks to 15 ml conical tubes, adding 12 mls HBSS,resuspending the cells and centrifuging down. The supernatant was thenpoured off ˜200 μls. To this was added 100 uCi ⁵¹Cr/1×10⁶ cells. Targetswere then incubated at 37° C., 7% CO₂ for 1 hr, then washed 2×12 mlHBSS.

Targets were resuspended in AIM-V with 5% human AB serum and added towells of 96-well U-bottom plates. Next, 1-20 ug/ml of peptides waspulsed onto targets at various time points dependent on the experiment;overnight, prior to the addition of ⁵¹Cr or following the ⁵¹Crincubation. CD8⁺ effector cells were added in AIM-V with 5% human ABserum. E:T ratios ranging from 5:1 to 50:1. Samples were in triplicate,200 μl final volume. Controls included spontaneous release of ⁵¹Cr intothe media, maximum release (incubation with 5% triton X100) andun-pulsed targets. The plates were spun at 700 RPM/5 minutes andincubated 4-6 hrs 37° C., 7% CO₂. The supernatant was harvested and ⁵¹Crrelease measured via gamma counter.

Cytokine ELISA Assays Measuring GM-CSF or γIFN Release

The Pharmingen (San Diego, Calif.) kits used may vary, need to followmanufacturer's instructions. In general, the protocol was as follows:

On Day 1, ELISA plates are coated with 100 μl diluted capture antibodyper well in coating buffer (0.1M Carbonate, pH 9.5). The plates are thensealed and wrapped with foil and incubated overnight at 4° C. Targetsare then incubated overnight with CD8⁺ cells, in 96 well U-bottomplates, in AIM-V media with 5% human AB serum. Samples in triplicate.

On Day 2, the wells of the ELISA plates were aspirated and washed 3×(Bio-Tek plate washer, Winooski, Vt.) with 300 μl/well wash buffer (PBS,0.05% TWEEN™ 20). The plates were then blocked with 200 μl/well assaydiluent, incubated at room temp for 1 hr, and the plates washed 3× withwash buffer.

100 μl standards or culture supernatant from each unknown sample wereadded to their respective wells and incubated at room temp for 2 hrs.

Plates were washed 5× with wash buffer, 100 μl working detector added toeach well, and the plates incubated at room temp for 1 hr. The plateswere then washed 7× with wash buffer, 100 μl substrate solution added toeach well, and the plates incubated for 15-30 min at room temp in dark.50 μl 2N H₂SO₄ was added to each well to stop the reaction. The plateswere then read at 450 nm within 30 minutes with λ correction 570 nm.

Example 9 C35 Peptide Mediated T Cell Lysis

T cell activity on EBV cells pulsed with C35 peptide epitopes wasanalyzed. Specifically, T cells obtained from two separate human donorsexpressing different HLA specificities were incubated with EBV-B targetcells pulsed with C35 peptide epitopes. The results are shown in Table10. The amino acid positions of the peptides in Table 10 refer to SEQ IDNO:2.

TABLE 10 T cell donor SB, HLA haplotype: A2, A3; B18, B44% Lysis EBV-B cell Target T cell clone No peptide +p72-86 +p77-85 #464.6 78.5 97.1 % Lysis EBV-B cell Target T cell clone No peptide +p17-31+p22-30 #72 0 0.5 72.8 #75 1.6 1.2 56.8 #104 0 6.4 100.3peptide 17-31 VEPGSGVRIVVEYAE peptide 22-30 GVRIVVEYApeptide 72-86 QLVFSKLENGGFPYE peptide 77-85 KLENGGFPYT cell donor LE, HLA haplotype: A3, A66; B8, B41% Lysis EBV-B cell Target T cell clone No peptide +p67-75 +p81-89 A 10.454.9 B 3.2 40.3 peptide 67-75 IEINGQLVF peptide 81-89 GGFPYEKDLT cell donor AH, HLA haplotype: A2, A11; B8, B35See induction of GM-CSF secretion by Line 4 T cells stimulated with:peptides 9-17 (SVAPPPEEV); 77-85 (KLENGGFPY); 104-112(KITNSRPPC); 105-113 (ITNSRPPCV)

As shown in TABLE 10, T cell clones from the donor expressing the A2,A3, B18, and B44 HLA haplotypes stimulated against the 15mercorresponding to amino acid residues 72-86 of C35 (SEQ ID NO:2) or the9mer corresponding to amino acids 77-85 (of SEQ ID NO:2) showed minimallytic activity on EBV-B target cells that had not been pulsed witheither peptide. However, a high level of lytic activity was observedwith the EBV-B cells pulsed with either peptide (78.5% and 97.1% forpeptide 72-86 and peptide 77-85, respectively). Similarly, T cells fromthe same donor raised against the 9 mer corresponding to amino acids22-30 of C35 (SEQ ID NO:2) did not lyse target cells not pulsed with thepeptide, but were very active in lysing target cells pulsed with thepeptide. Notably, however, target cells pulsed with the 15mercorresponding to amino acids 17-31 of C35 (SEQ ID NO:2) exhibitedminimal lysis even though it contains the 9mer 22-30 (of SEQ ID NO:2). Tcell clones from another donor expressing the A3, A66, B8 and B41 HLAhaplotypes stimulated against the 9mers corresponding to amino acids67-75 and 81-89 of C35 (SEQ ID NO:2) exhibited moderate lytic activity(54.9% and 40.3%, respectively) on EBV-B target cells pulsed with thesepeptides.

The high level of lytic activity observed with EBV-B cells pulsed witheither the 9mer p77-85 (of SEQ ID NO:2) or the 15mer p72-86 (of SEQ IDNO:2) demonstrates that in some instances larger peptides comprising aC35 peptide epitope are effective in stimulating a T cell responsesimilar to that achieved with the actual epitope. However, the fact thatminimal lysis was observed with p17-31 even though it contained a C35peptide epitope, p22-30 (of SEQ ID NO:2), that triggered significantlytic activity, demonstrates that this effect is not seen in everyinstance.

Example 10 Monoclonal Antibodies Specific for C35

Immunization Protocols:

In order to generate monoclonal antibodies specific for C35, BALB/cByJmice were immunized using one of three methods.

1. A syngeneic mouse fibrosarcoma cell line, BCA34, was transduced toexpress human C35 by infecting cells with C35 recombinant retrovirus.Stable C35 expressing cells were selected for resistance to G418. Twomillion cells were injected per mouse, and spleens were removed 23 dayslater for fusion with NS1 mouse myeloma cells, employing methods wellknown to those practiced in the art.

2. Line1, a poorly immunogenic mouse small cell lung carcinoma, wastransduced to express C35 as described above for BCA34. Mice wereimmunized with 20,000 cells, and spleens were removed 20 days later forfusion with NS1 mouse myeloma cells.

3. Mice received an intraperitoneal injection of 10 million pfu of avaccinia virus recombinant for human C35, VV.hC35. Spleens were removedafter 15 days for fusion with NS1 mouse myeloma cells.

Screening Hybridoma Clones by ELISA.

ELISA assay were used to screen antibodies secreted by 500-1000hybridoma clones from each fusion for reactivity and specificity forC35. ELISA plates were coated with C35 protein or Beta galactosidase asa negative control. Both proteins were generated and purified from E.coli in a similar fashion, including cleavage of selection tags.Supernatants from hybridoma clones were then incubated on the coatedplates, followed by incubation with anti-mouse antibodies labeled withhorseradish peroxidase (HRP), and detection with OPD substrate. Clonesthat reacted strongly with C35 protein but did not react withβ-galactosidase were selected for further characterization. Eachselected hybridoma clone was subcloned several times to develop a stableantibody-producing cell line. Table 11 lists the clones with confirmedspecificity for C35. In several cases, antibody specificity wasconfirmed by Western blot of a C35 protein gel or by immunohistochemicalstaining as described below. Immunoglobulin heavy and light chain geneswere cloned and sequenced from three C35 specific hybridomas of whichtwo were determined to be identical.

TABLE 11 Hybridoma clones with demonstrated specificity for C35 VariableCLONE Immunization Isotype Reactivity region genes 1B2 BCA34.hC35 IgM1B3* BCA34.hC35 IgG1 Western 1E 11 BCA34.hC35 IgG1 cloned (same as 1F2)1F2 BCA34.hC35 IgG1 Western, IHC cloned (same as 1E11) 3E 9 BCA34.hC353E 10* BCA34.hC35 IgG1 Western KC5 Line1.hC35 IgM 11B10 VV.hC35 IgMWestern cloned *antibody 1B3 is identical in sequence to antibody 3E10

FIG. 19 shows a representative ELISA experiment. All clones were testedin triplicate on at least 3 separate occasions.

III. Western Blot Immunodetection

Western Blot Immunodetection was performed with supernatants fromselected hybridoma clones. Antibodies from 4 hybridomas (1B3, 1F2, 3E10,11B10) reacted specifically with hC35 protein in this assay. In theseexperiments, 100 ng of purified recombinant C35 protein or controlβ-galactosidase protein was loaded in each lane of an 18% SDSpolyacrylamide gel. Gels were transferred to PVDF membrane, as is wellknown to those practiced in the art. Membranes were blocked with Trisbuffered saline (TBS), including Tween-20 and 5% non-fat dry milk. Eachblot was incubated with various dilutions of hybridoma supernatants asthe primary antibody, followed by incubation with a secondary antibody,goat anti-mouse IgG conjugated to HRP, and detected with thechemiluminescent substrate, ECL. Results for antibodies 1B3, 1F2, and3E10 are shown in FIG. 20. Similar results were obtained with 11B10 (notshown).

IV. Immunohistochemistry.

ELISA and Western Blot data show that these monoclonal antibodies reactwith recombinant C35 protein. In addition, monoclonal antibody 1F2 wasshown to have utility for immunohistochemical staining of primary breasttumor sections. FIG. 21 demonstrates that monoclonal antibody 1F2 candetect high levels of endogenous C35 expression in human breast tumors,with little or no staining of normal breast tissue. Antibody wasaffinity purified from 1F2 hybridoma supernatant. Paraffin-embeddedslides were deparaffinized with xylene and rehydrated through gradedalcohols. Target retrieval was achieved through steam and high pHtreatment. Sections were blocked with normal serum, incubated withaffinity purified 1F2 (5 mg/ml), followed by Vector ABC universaldetection kit and development with DAB substrate. Slides werecounterstained with hematoxylin, dehydrated with EtOH/xylene, andcoverslipped.

FIG. 21 demonstrates strong staining of a section of invasive breastadenocarcinoma from patient 01A6, while normal breast tissue from thesame patient is negative. Staining with the antibody could be competedwith soluble C35 protein, but not with the irrelevant proteinβ-galactosidase (data not shown), this demonstrates that staining isspecific for C35.

V. Cloning Variable Region Genes of Monoclonal Antibodies

Once specificity of individual clones was confirmed, cell pellets of thehybridoma clones and the fusion partner NS1 were snap frozen at −80° C.for later cloning of variable region genes. RNA was extracted from thecell pellets and full-length cDNA was generated using Invitrogen'sGENERACER™ Kit. Briefly, the 5′ end of the RNA is decapped, then ligatedto GENERACER™ 5′ oligo. Reverse strand cDNA is generated using oligo-dTprimers and reverse transcriptase. Double stranded cDNA was amplified byPCR using gene racer oligo as the 5′ primer and a 3′ primer designedfrom a conserved sequence in the IgG1 constant region. PCR products werecloned and sequenced. Clones were identified that had unique sequenceswhen compared to those of the fusion partner. The sequences weresubmitted to IgBLAST on the NCBI database to map the variable regionframework and complementarity determining regions (CDR). To confirm thatthese V-genes are specific for C35, recombinant antibodies encodingthese sequences have been generated and expressed in vitro and assayedby ELISA and Western (data not shown).

The sequences of two unique monoclonal antibody V-genes are shown below.Clones 1F2 and 1E11 have identical V-genes. Bold indicates CDR regions.Underline indicates framework regions. 1E11/1F2 Kappa and IgG1 V-genes(SEQ ID NOs:148 and 149); 3E10 Kappa and IgG1 V-genes (SEQ ID NOs:150and 151).

1E11/1F2 V-genes: Kappaatggattttcaggtgcagattttcagcttcctgctaatcagtgcctcagtcagaatgtccagaggacaaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatatcctgcagtgccagctcaagtgt aagttacatgaactggtaccagcagaagccaggatcctcccccaaaccctggatttat cacacatccaacct ggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgc cacttattactgccaacagtatcatagttacccacccacgttcggaggggggaccaa gctggaaataaaa IgG1atgaaagtgttgagtctgttgtacctgttgacagccattcctggtatcctgtctgatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtcactggctactccatcaccagtggttattt ctggaac tggatccggcagtttccagggaacaaactggaatggatgggctacataagctacgacggtagca ataactccaacccatctctcaaaaatcgaatctccttcactcgtgacacatctaagaaccagtttttcctgaagtttaattctgtgactactgacgactcagctgcatattactgtacaaga ggaactacggggtttgcttactggggcca agggactctggtcactgtctctgca 3E10 V-genes: KAPPAAtgaggttccaggttcaggttctggggctccttctgctctggatatcaggtgcccactgtgatgtccagataacccagtctccatcttttcttgctgcatctcctggagaaaccattactattaattgcagggcaagtaagtacattagcaa acatttagtctggtatcaggagaaacctggagaaactaaaaagcttcttatctac tctggatccactttgcaatc tggacttccatcaaggttcagtggcagtggatctggtacagatttcactctcaccatcagtagcctggagcctgaagattttgcaatgtattactgt caacagcataatgaatacccgctcacgttcggtgctgggaccaagctggagct gaaa IgG1 atgatggtgttaagtcttctgtacctgttgacagcccttccgggtatcctgtcagaggtgcagcttcaggagtcaggacctagcctcgtgaaaccttctcagactctgtccctcacctgttctgtcactggcgactccatcaccagtggttactggaac tggatccggaaattcccaggaaataaacttgaatacgtggggtacataagctacagtggtggcac ttactacaatccatctctcaaaagtcgaatctccatcactcgagacacatccaagaaccactactacctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagaggtgcttactacgggggggccttttttc cttacttcgatgtctggggcgctgggaccacggtcaccgtctcctca

C35 is an Oncogene for Normal Breast Epithelial Cells

C35 is a gene of unknown function that we have previously shown isoverexpressed in a large fraction of human breast and bladder tumors.Antibody to C35 inhibits growth of C35 positive tumor cells in vitro.This suggests that the C35 gene product may play a role in signaltransduction on the tumor cell membrane and raises the possibility thatC35 is an oncogene. Growth of colonies in soft agar is an acceptedmeasure of anchorage independence, a property that distinguishes cellsthat have undergone tumor transformation from normal cells. In order toassay the oncogenic activity of C35, a line of immortalized,non-tumorigenic breast epithelial cells (H16N2) was transected withpTag.hC35 recombinant for the full length C35 gene, with empty pTagvector alone (pCMV-tag mammalian expression vector, StratageneCorporation, LaJolla, Calif.) or with ras, a known oncogene. Formationof colonies in soft agar, indicative of transformation, was assessed andcompared to 21-MT1 breast tumor cells (see Table 12 below). The resultsshow a factor of 10 increase in the number of soft agar colonies formedfollowing transfection with C35. This is comparable to the frequency ofcolonies formed following transfection with ras or that result when the21-MT1 tumor line is plated in soft agar.

Transformed colonies were picked from soft agar and it was attempted topropagate them in liquid medium in 24-well plates. The small number ofapparent colonies recovered following transfection with the vectorcontrol could not be successfully propagated in liquid culture. Incontrast, colonies recovered following transfection with C35 or with rashave been successfully established as cell lines. It appears that eventhe small number of control colonies recovered from soft agar representabortive growth.

TABLE 12 C35 Transforms Human Breast Epithelial Cells Cell Line # ofcolonies Propagated? H16N2.pTag (vector control) 14 No H16N2.pTag-hC35150 Yes H16N2.ras (positive control) 250 Yes 21-MT1 (metastatic tumorline, C35⁺) 94 Yes

The fact that C35 appears to contribute to the transformed phenotype oftumor cells enhances the potential utility of a C35-based cancer vaccineor C35-specific antibodies as therapeutic agents for breast and bladdercancer. Immune evasion by down regulation of C35 gene expression intumor cells or down regulation of C35 expression at the tumor surfacemembrane is less likely to be an obstacle to successful C35-basedimmunotherapy if the C35 gene product is required to maintain the tumorphenotype.

Example 12 Fluorescence Polarization to Monitor Mhc-Peptide Interactionsin Solution

Fluorescence is characterized by a process of absorption of incidentradiation at one wavelength, followed by the emission of radiation atanother wavelength. This behavior was first described by G. G. Stokes(1852) in the form of Stokes' Law of Fluorescence in which he statedthat fluorescence (emission) always appears at a wavelength greater thanthe wavelength of the incident (excitation) radiation.

This behavior was successfully explained by A. Einstein (1905) usingPlanck's quantum hypothesis. A quantum of incident light with an energyof Eexcit is absorbed by the fluorescent molecule raising its energy toEexcit. This is quickly followed by a downward transition of themolecule to one of the vibration levels in the ground state, Eemiss,with the emission of a quantum of light.

By using a fluorescent dye to label a small molecule, its binding toanother molecule of equal or greater size can be monitored usingfluorescence polarization (FP). FP operates on the principle that smallmolecules rotate faster than large molecules. If a molecule is labeledwith a fluorophore, this rotation can be measured by exciting thefluorophore with plane polarized light and then measuring the emittedlight with polarizers parallel and perpendicular to the plane ofexcitation to determine if it is still oriented in the same plane as itwas when excited. If a fluorophore is labeled on a small molecule itwill rotate in the time between excitation and emission and the lightemitted will be depolarized. If the labeled molecule binds to a largemolecule (effectively increasing its overall size) the molecule will notrotate in the time between excitation and emission, and the lightemitted will be polarized resulting in a polarization change between thefree and bound forms. For convenience, units are usually 1000 mP=P

The kinetics of association between HLA-A*0201 and a specificfluorescent-labeled peptide was determined in the presence of a testcompetitor peptide at 100 μM. Binding increases over time until itplateaus when specific binding reaches equilibrium at Ymax. If the addedtest-competitor is able to compete, equilibrium will be reached fasterbut with less binding. Ymax can be used to calculate % inhibition if thesystem is calibrated with an irrelevant competitor (0%) and a relevantcompetitor (100%). In this case, the irrelevant competitor was theHLA-B*2705 binding (non-HLA-A*0201 binding) peptide GRAFVTIGK (SEQ IDNO:2128) while the relevant competitor was the known HLA-A*0201 bindingpeptide KLGEFYNQMM (SEQ ID NO:2129). To obtain Ymax, the curves arefitted to a mono-exponential association model using non-linearregression.

The following eight peptides (based on amino acid sequence of SEQ IDNO:2 were found to have High to Medium binding capacity to HLA-A*0201 asreflected by relative inhibition of binding of the relevant competitor.

Peptide % inhibition S9 to V17 96.9 I25 to A33 94.9 S21 to Y29 90.8 I105to V113 86.4 F65 to L73 74.8 G22 to A30 67 T38 to V46 47.3 G61 to I6946.5

A similar analysis was conducted with C35 peptides that bind to solubleHLA-B*0702 employing a fluorescent labeled HLA-B*0702 binding standardand suitable relevant and irrelevant peptides to calibrate inhibition ofbinding by test peptides. Note that these inhibition values aresensitive to the choice and concentration of both MHC molecule andspecific fluorescent peptides under the same assay conditions. It is notmeaningful, however, to compare HLA-A*0201 and HLA-B*0702 inhibitionvalues. As shown below, six C35-derived peptides nine amino acids inlength were found to have high or medium relative binding affinity toHLA-B*0702.

Peptide % Inhibition K104 to A112 69.4 (analog with Ala substituted forCys at 112) N107 to L115 68.2 (analog with Ala substituted for Cys at112) E4 to P12 54.6 G63 to G71 51.4 I105 to V113 45.2 (analog with Alasubstituted for Cys at 112) T62 to N70 38.8

Example 13 Construction of NTerminal and/or CTerminal Deletion Mutants

The following general approach may be used to clone a Nterminal orCterminal deletion C35 deletion mutant. Generally, two oligonucleotideprimers of about 1525 nucleotides are derived from the desired 5′ and 3′positions of a polynucleotide of SEQ ID NO: 1. The 5′ and 3′ positionsof the primers are determined based on the desired C35 polynucleotidefragment. An initiation and stop codon are added to the 5′ and 3′primers respectively, if necessary, to express the C35 polypeptidefragment encoded by the polynucleotide fragment. Preferred C35polynucleotide fragments are those encoding the candidate MHC class Iand MHC class II binding peptides disclosed above in the “Polynucleotideand Polypeptide Fragments” section of the Specification.

Additional nucleotides containing restriction sites to facilitatecloning of the C35 polynucleotide fragment in a desired vector may alsobe added to the 5′ and 3′ primer sequences. The C35 polynucleotidefragment is amplified from genomic DNA or from the cDNA clone using theappropriate PCR oligonucleotide primers and conditions discussed hereinor known in the art. The C35 polypeptide fragments encoded by the C35polynucleotide fragments of the present invention may be expressed andpurified in the same general manner as the full length polypeptides,although routine modifications may be necessary due to the differencesin chemical and physical properties between a particular fragment andfull length polypeptide.

As a means of exemplifying but not limiting the present invention, thepolynucleotide encoding the C35 polypeptide fragment is amplified andcloned as follows: A 5′ primer is generated comprising a restrictionenzyme site followed by an initiation codon in frame with thepolynucleotide sequence encoding the Nterminal portion of an MHC bindingpeptide epitope listed in any of Tables 1 through 6. A complementary 3′primer is generated comprising a restriction enzyme site followed by astop codon in frame with the polynucleotide sequence encoding Cterminalportion of a C35 MHC binding peptide epitope listed in any of Tables 1through 6.

The amplified polynucleotide fragment and the expression vector aredigested with restriction enzymes which recognize the sites in theprimers. The digested polynucleotides are then ligated together. The C35polynucleotide fragment is inserted into the restricted expressionvector, preferably in a manner which places the C35 polypeptide fragmentcoding region downstream from the promoter. The ligation mixture istransformed into competent E. coli cells using standard procedures andas described in the Examples herein. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

Example 14 Protein Fusions of C35

C35 polypeptides are preferably fused to other proteins. These fusionproteins can be used for a variety of applications. For example, fusionof C35 polypeptides to Histag, HAtag, protein A, IgG domains, andmaltose binding protein facilitates purification. (See Example 5; seealso EP A 394,827; Traunecker et al., Nature 331:8486 (1988).)Similarly, fusion to IgG1, IgG3, and albumin increases the halflife timein vivo. Nuclear localization signals fused to C35 polypeptides cantarget the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenonfused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portioncan be ligated into the BamHI cloning site. Note that the 3′ BamHI siteshould be destroyed. Next, the vector containing the human Fc portion isrerestricted with BamHI, linearizing the vector, and C35 polynucleotide,isolated by the PCR protocol described in Example 1, is ligated intothis BamHI site. Note that the C35 polynucleotide is cloned without astop codon, otherwise a fusion protein will not be produced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc region: (SEQ ID NO: [84])GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGCGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGATGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAG GAT

A preferred fusion product is fusion of a C35 peptide to the aminoterminus of an MHC molecule in such fashion that the peptide willnaturally occupy the MHC peptide binding groove. Kang, X. et al., CancerRes. 57:2025 (1997) have reported that such fusion proteins can beemployed in vaccine compositions that are especially effective forstimulation of specific T cells.

Example 15 Method of Detecting Abnormal Levels of C35 in a BiologicalSample

C35 polypeptides can be detected in a biological sample, and if anincreased or decreased level of C35 is detected, this polypeptide is amarker for a particular phenotype. Methods of detection are numerous,and thus, it is understood that one skilled in the art can modify thefollowing assay to fit their particular needs.

For example, antibodysandwich ELISAs are used to detect C35 in a sample,preferably a biological sample. Wells of a microtiter plate are coatedwith specific antibodies to C35, at a final concentration of 0.2 to 10ug/ml. The antibodies are either monoclonal or polyclonal. The wells areblocked so that nonspecific binding of C35 to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining C35. Preferably, serial dilutions of the sample should beused to validate results. The plates are then washed three times withsaline to remove unbounded C35.

Next, 50 ul of specific antibodyalkaline phosphatase conjugate thatrecognizes a C35 antigenic determinant which does not overlap with thatrecognized by the plate bound antibody, at a concentration of 25400 ng,is added and incubated for 2 hours at room temperature. The plates areagain washed three times with deionized or distilled water to removeunbounded conjugate.

Add 75 ul of 4methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot C35 polypeptide concentration on the Xaxis (log scale) andfluorescence or absorbance on the Yaxis (linear scale). Interpolate theconcentration of the C35 in the sample using the standard curve.

Example 16 Formulating a Polypeptide

The C35 composition will be formulated and dosed in a fashion consistentwith good medical practice, taking into account the clinical conditionof the individual patient (especially the side effects of treatment withthe C35 polypeptide alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofC35 administered parenterally per dose will be in the range of about 1ug/kg/day to 10 mg/kg/day of patient body weight, although, as notedabove, this will be subject to therapeutic discretion. More preferably,this dose is at least 0.01 mg/kg/day, and most preferably for humansbetween about 0.01 and 1 mg/kg/day. If given continuously, C35 istypically administered at a dose rate of about 1 ug/kg/hour to about 50ug/kg/hour, either by 14 injections per day or by continuoussubcutaneous infusions, for example, using a minipump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

Pharmaceutical compositions containing C35 are administered orally,rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), bucally, or as an oral or nasal spray.“Pharmaceutically acceptable carrier” refers to a nontoxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

C35 is also suitably administered by sustainedrelease systems. Suitableexamples of sustainedrelease compositions include semipermeable polymermatrices in the form of shaped articles, e.g., films, or microcapsules.Sustainedrelease matrices include polylactides (U.S. Pat. No. 3,773,919,EP 58,481), copolymers of Lglutamic acid and gammaethylLglutamate(Sidman, U. et al., Biopolymers 22:547556 (1983)), poly (2 hydroxyethylmethacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167277(1981), and R. Langer, Chem. Tech. 12:98105 (1982)), ethylene vinylacetate (R. Langer et al.) or polyD( )3hydroxybutyric acid (EP 133,988).Sustainedrelease compositions also include liposomally entrapped C35polypeptides. Liposomes containing the C35 are prepared by methods knownper se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA82:36883692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:40304034(1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;Japanese Pat. Appl. 83118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. Ordinarily, the liposomes are of the small (about 200800Angstroms) unilamellar type in which the lipid content is greater thanabout 30 mol. percent cholesterol, the selected proportion beingadjusted for the optimal secreted polypeptide therapy.

For parenteral administration, in one embodiment, C35 is formulatedgenerally by mixing it at the desired degree of purity, in a unit dosageinjectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is nontoxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptide.

Generally, the formulations are prepared by contacting C35 uniformly andintimately with liquid carriers or finely divided solid carriers orboth. Then, if necessary, the product is shaped into the desiredformulation. Preferably the carrier is a parenteral carrier, morepreferably a solution that is isotonic with the blood of the recipient.Examples of such carrier vehicles include water, saline, Ringer'ssolution, and dextrose solution. Nonaqueous vehicles such as fixed oilsand ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

C35 is typically formulated in such vehicles at a concentration of about0.1 mg/ml to 100 mg/ml, preferably 110 mg/ml, at a pH of about 3 to 8.It will be understood that the use of certain of the foregoingexcipients, carriers, or stabilizers will result in the formation ofpolypeptide salts.

C35 used for therapeutic administration can be sterile. Sterility isreadily accomplished by filtration through sterile filtration membranes(e.g., 0.2 micron membranes). Therapeutic polypeptide compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

C35 polypeptides ordinarily will be stored in unit or multidosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10 ml vials are filled with 5 mlof sterilefiltered 1% (w/v) aqueous C35 polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized C35 polypeptide using bacteriostaticWaterforInjection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, C35 maybe employed in conjunction with other therapeutic compounds.

Example 17 C35 Peptide Mediated Cell Lysis

T cell activity on various cell lines was analyzed. Specifically, Tcells obtained from one human donor stimulated with peptide pulsed DCswere incubated with different cell line targets. The results are shownin Tables 13 and 14.

CD8+ cells were stimulated using DCs and pulsed autologous DCs asdescribed in Example 8. DCs were pulsed with a K104-V113 peptidefragment of C35 (SEQ ID NO:2) with TCEP (K104-V113), a reducing agent toprotect the cysteine contained in the peptide. DCs were also pulsed witha K104-V113 peptide fragment containing an alanine or serinesubstitution at amino acid position 112 (K104-V113-Ala (SEQ ID NO:176)and K104-V113-Ser (SEQ ID NO:178)). DCs were also pulsed with aK104-V113 peptide fragment of C35 containing a cysteinylated cysteine atamino acid position 112 (K104-V113-cys-cys (SEQ ID NO: 2130).

The target cells used in the experiment were as follows: a normal breastendothelial cell line which is HLA-A2 positive and has low levels of C35expression (H16.A2); a breast tumor cell line which is HLA-A2 negativeand has high levels of C35 expression (21NT); a breast tumor cell linewhich is HLA-A2 positive and has high levels of C35 expression(21NT.A2); a cell line which is sensitive to non-specific killing by NKcells (K562); a head and neck cancer cell line which is HLA-A2 positiveand does not express C35 (PCl-13); and the PCl-13 cell line pulsed witha 104-113 peptide fragment of C35 with a serine substitution at aminoacid position 112 (SEQ ID NO:178) (PCl-13 K104-V113-Ser).

Cell lysis was measured in a cytotoxicity 51Cr release assay asdescribed in Example 8. Raw chromium values, as measured from thesupernatant from target cells used in the experiments, are shown inTable 13. The spontaneous values are from target cells incubated inmedium with no CD8+ cells added. The maximum values are from targetcells incubated in HCl so that all cells are lysed. The percentage ofcell lysed data is shown in Table 14. The peptides in Tables 13-16 havethe following SEQ ID NOs: K104-V113-Ala (SEQ ID NO:176), K104-V113-Ser(SEQ ID NO:178), K104-V113-cys-cys (SEQ ID NO:2130), and K104-V113 (ofSEQ ID NO:2).

TABLE 13 T cell donor SB, HLA haplotype: A2, A3; B18, B44 Raw ChromiumValues PCl-13 In vitro Stimulus H16.A2 21NT 21NT.A2 K562 PCl-13K104-V113-Ser DC 466 177 732 453 691 673 K104-V113-Ala 479 155 763 246468 746 K104-V113-Ser 584 185 3228 323 581 2588 K104-V113- 440 171 1329287 486 1772 cys-cys K104-V113 483 137 1429 311 804 1403 Spontaneous 425173 440 267 543 538 Maximum 6628 2654 4853 2481 3962 4029 % Spontaneous6.4 6.5 9.1 10.8 13.7 13.4

TABLE 14 T cell donor SB, HLA haplotype: A2, A3; B18, B44 Percentage ofTarget Cells Lysed Pcl-13 In vitro Stimulus H16.A2 21NT 21NT.A2 K562PCl-13 K104-V113-Ser DC 0.7 0.2 6.6 8.4 4.3 3.9 K104-V113-Ala 0.9 −0.77.3 −0.9 −2.2 6.0 K104-V113-Ser 2.6 0.5 63.2 2.5 1.1 58.7 K104-V113- 0.2−0.1 20.1 0.9 −1.7 35.3 cys-cys K104-V113 0.9 −1.5 22.4 2.0 7.6 24.8

The cytotoxicity 51Cr release assay was repeated with the same CD8+cells which received in vitro stimulus using C35 peptides describedabove and in Example 8. In addition to the cell lines described above,the 21NT.A2 breast tumor cell line pulsed with a K104-V113 C35 peptidefragment containing a serine substitution at amino acid position 112(21NT.A2-Ser (SEQ ID NO:178)) was used. Various melanoma cell lines werealso used as target cells. Melanoma cell lines 1700 (Mel 1700), 501 (Mel501) and F002 (Mel F002) are HLA-A2 positive and express C35. Melanomacell line 1359 (Mel 1359) is HLA-A2 negative and expresses C35. Theeffector to target cell ratio was 30:1 for this experiment. CD8+ cellsused in the experiment have been maintained in IL-2 for 12 days afterthe fourth stimulation. Raw chromium values, as measured from thesupernatant from target cells used in the experiments, are shown inTable 15. The percentage of lysed cells is shown in Table 16.

TABLE 15 T cell donor SB, HLA haplotype: A2, A3; B18, B44 Raw ChromiumValues In vitro Stimulus H16.A2 21NT.A2 21NT.A2-Ser K562 DC 916 23832470 1403 K104-V113-Ala 829 1365 2423 815 K104-V113-Ser 1018 4957 4762974 K104-V113-cys-cys 1024 2337 4474 1713 K104-V113 948 2753 3729 1156Spontaneous 729 875 855 619 Maximum 5848 8521 7830 7356 In vitroStimulus Mel 1700 Mel 1359 Mel 501 Mel F002 DC 1743 2443 2470 1403K104-V113-Ala 1543 3050 2423 815 K104-V113-Ser 2951 2357 4762 974K104-V113-cys-cys 1857 2299 4474 1713 K104-V113 1964 2758 3729 1156Spontaneous 1387 2050 1637 1716 Maximum 7887 12157 8953 8003

TABLE 16 T cell donor SB, HLA haplotype: A2, A3; B18, B44 Percentage ofTarget Cells Lysed In vitro Stimulus H16.A2 21NT.A2 21NT.A2-Ser K562 DC3.7 19.7 23.2 11.6 K104-V113-Ala 2.0 6.4 22.5 2.9 K104-V113-Ser 5.6 53.456.0 5.3 K104-V113- 5.8 19.1 51.9 16.2 cys-cys K104-V113 4.3 24.6 41.58.0 In vitro Stimulus Mel 1700 Mel 1359 Mel 501 Mel F002 DC 5.5 3.9 15.811.5 K104-V113-Ala 2.4 9.9 11.1 7.6 K104-V113-Ser 24.1 3.0 45.3 15.1K104-V113- 7.2 2.5 21.2 11.2 cys-cys K104-V113 8.9 7.0 36.0 15.7

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, Examples, and Sequence Li sting is hereby incorporatedherein by reference.

1. An isolated antibody or antigen binding fragment thereof comprising aheavy chain variable domain and a light chain variable domain, whereinsaid heavy chain variable domain comprises the same three CDRs encodedby a polynucleotide having the sequence of SEQ ID NO: 149; and whereinsaid light chain variable domain comprises the same three CDRs encodedby a polynucleotide having the sequence of SEQ ID NO: 148, and whereinthe antibody or antigen binding fragment thereof specifically binds C35.2. The antibody or antigen binding fragment thereof according to claim1, wherein the polynucleotides encoding the heavy chain variable domainCDRs of SEQ ID NO: 149 comprise: (a) nucleotides 145 to 162 of SEQ IDNO:149; (b) nucleotides 205 to 252 of SEQ ID NO:149; and (c) nucleotides349 to 369 of SEQ ID NO:149.
 3. The antibody or antigen binding fragmentthereof according to claim 1, wherein the polynucleotides encoding thelight chain variable domain CDRs of SEQ ID NO; 148 comprise: (a)nucleotides 136 to 165 of SEQ ID NO:148; (b) nucleotides 211 to 231 ofSEQ ID NO:148; and (c) nucleotides 328 to 354 of SEQ ID NO:148.
 4. Theantibody or antigen binding fragment thereof according to claim 1,wherein the polynucleotides encoding the heavy chain variable domainCDRs of SEQ ID NO: 149 and the light chain variable domain CDRs of SEQID NO: 148 comprise: (a) nucleotides 145 to 162 of SEQ ID NO:149; (b)nucleotides 205 to 252 of SEQ ID NO:149; (c) nucleotides 349 to 369 ofSEQ ID NO:149; (d) nucleotides 136 to 165 of SEQ ID NO:148; (e)nucleotides 211 to 231 of SEQ ID NO:148; and (f) nucleotides 328 to 354of SEQ ID NO:148.
 5. The antibody or antigen binding fragment accordingto claims 1, wherein said heavy chain variable domain is encoded by apolynucleotide comprising the sequence of SEQ ID NO:
 149. 6. Theantibody or antigen binding fragment thereof according to claim 1,wherein said light chain variable domain is encoded by a polynucleotidecomprising the sequence of SEQ ID NO:
 148. 7. The antibody or antigenbinding fragment thereof according to claim 1, wherein said antibody isa full antibody.
 8. The antibody or antigen binding fragment thereofaccording to claim 1, wherein said antigen binding fragment is selectedfrom the group consisting of: a Fab fragment, a F(ab′)2 fragment, and ascFv fragment.
 9. The antibody or antigen binding fragment thereofaccording to claim 1, wherein said antibody or antigen binding fragmentis chimeric.
 10. The antibody or antigen binding fragment thereof ofclaim 1, wherein said antibody or antigen binding fragment is humanized.11. A composition comprising the antibody or antigen binding fragmentthereof according to claim
 1. 12. The composition according to claim 11,wherein said composition further comprises a therapeutic agent.
 13. Thecomposition according to claim 12, wherein said therapeutic agent isselected from the group consisting of: an anti-tumor drug, a cytotoxin,and a radioactive agent.
 14. The composition according to claim 13,wherein said therapeutic agent is a cytotoxin.
 15. The compositionaccording to claim 14, wherein said cytotoxin is selected from the groupconsisting of: taxol, ricin, doxorubicin, cytochalasin B, gramicidin D,ethidium bromide, etoposide, tenoposide, colchicin, dihydroxy anthracindione, 1 dehydrotestosterone, and glucocorticoid.
 16. The compositionaccording to claim 12, wherein said therapeutic agent is conjugated toor complexed with said antibody or antigen binding fragment thereof. 17.A diagnostic kit for detecting the presence of C35 in a sample, said kitcomprising: (a) the antibody or antigen binding fragment thereofaccording to claim 1; and (b) a conjugate comprising a specific bindingpartner for the C35 specific antibody and a label capable of producing adetectable signal.
 18. The antibody or antigen binding fragment thereofaccording to claim 1, further comprising a human immunoglobulin heavychain constant region or fragment thereof.
 19. The antibody or antigenbinding fragment thereof according to claim 18, wherein said heavy chainconstant region is human IgG.
 20. The antibody or antigen bindingfragment thereof according to claim 1, further comprising a humanimmunoglobulin light chain constant region.
 21. The antibody or antigenbinding fragment thereof according to claim 20, wherein said light chainconstant region is human kappa.